Proteins

ABSTRACT

An improved dough for preparing bakery products is made by substituting a bacterial xylanase for the usual fungal xylanase, resulting in a dough which is less sticky. Suitable bacterial xylanases and xylanase inhibitors are identified.

BACKGROUND OF THE PRESENT INVENTION

The present invention relates to proteins.

In particular, the present invention relates to the isolation of andcharacterisation of an endogenous endo-β-1,4-xylanase inhibitor that ispresent in wheat flour and its effect on different xylanases. Thepresent invention also relates to xylanases identified by a screen usingthe inhibitor and to novel xylanases identified thereby.

BACKGROUND ART

Xylanases have been used in bakery for several years.

In this regard, it is known that wheat flour contains arabinoxylanoriginating from the endosperm cell walls. The amount of arabinoxylan inthe flour differs depending on the origin of the flour—for example, seeRouau et al, Journal of Cereal Science (1994), 19, 259-272 Effect of anEnzyme Preparation Containing Pentosanases on the Bread-making Qualityof Flour in Relation to Changes in Pentosan Properties; Fincher andStone, (1986) Advances in Cereal Technology, Vol. VIII (Why Pomeranz,Ed.) AACC, St Paul, Minn., 207-295; and Meuser and Suckow (1986),Chemistry and Physics of Baking (J. M. V. Blanchard, P J Frasier and TGillard, Eds.) Royal Society of Chemistry, London, 42-61. Typically theamount of arabinoxylan can vary from 2-5% ((w/w) based on flour dryweight).

Fincher and Stone (1986) report 70% of the polysaccharides in theendosperm cell wall are arabinoxylan. A characteristic feature ofarabinoxylan is its ability to bind to water. Part of the arabinoxylanis water insoluble pentosan (WIP) and part is water soluble pentosan(WSP). Experimental results have shown a correlation between degradationof WIP to high molecular weight (HMW) water soluble polymers and breadvolume.

During the production of a bakery product, it is known that using axylanase at a proper dosage may result in a more stable dough system(which will typically comprise salt, flour, yeast and water) and abetter volume of, for example, raised bread.

In this respect, a good xylanase for increasing bread volume shouldsolubilise WIP giving an increased viscosity in the dough liquid withoutfurther degradation of WSP into xylose oligomers. This degradation ofWIP into low molecular weight (LMW) WSP is believed to be detrimentalfor the dough properties and may give rise to stickiness (Rouau et aland McCleary (1986) International Journal of Biological Macro Molecules,8, 349-354).

U.S. Pat. No. 5,306,633 discloses a xylanase obtained from a Bacillussubtilis strain. Apparently, this xylanase may improve the consistencyand increase the volume of bread and baked goods containing the same.

Another xylanase from Bacillus subtilis has been isolated and sequenced(see Paice, M. G., Bourbonnais, R., Desrochers, M., Jurasek, L. andYaguchi, M. A xylanase gene from Bacillus subtilis: nucleotide sequenceand comparison with B. pumilus gene, Arch. Microbiol. 144, 201-206(1986)).

It has been considered for some time now that bacterial xylanases wouldproduce very sticky dough. Hence, one would normally expect thexylanases of Bacillus subtilis—such as that of U.S. Pat. No.5,306,633—to produce a very sticky dough.

Prior art enzymes which caused stickiness had to be used in carefullycontrolled amounts so that stickiness would not adversely affecthandling to such a degree that effective commercial handling washampered. However, the need to carefully control dosage prohibited theaddition of xylanase directly to flour prior to production of the dough.It was therefore necessary with prior art systems to add the xylanase ina very controlled manner during the production of the dough.

To date, fungal xylanases have been typically used in baking. Forexample, J Maat et al. (Xylans and Xylanases, edited by J Visser et al,349-360, Xylanases and their application in bakery) teach aβ-1,4-xylanase produced by an Aspergillus Niger var. awarmori strain.According to these authors, the fungal xylanase is effective inincreasing the specific volume of breads, without giving rise to anegative side effect on dough handling (stickiness of the dough) as canbe observed with xylanases derived from other fungal or from bacterialsources.

It has been proposed by W Debyser et al., (J. Am. Soc. Brew. Chem.55(4), 153-156, 1997, Arabinoxylan Solubilization and Inhibition of theBarely Malt Xylanolytic System by Wheat During Mashing with WheatWholemeal Adjunt: Evidence for a New Class of Enzyme Inhibitors inWheat), that xylanase inhibitors may be present in wheat. The inhibitordiscussed by W Debyser et al. was not isolated. Furthermore, it is notdisclosed by W Debyser et al. whether the inhibitor is endogenous ormicrobiological. Moreover, no chemical data were presented for thisinhibitor.

The presence of xylanase inhibitor in wheat flour has also recently beendiscussed by X Rouau and A Surget, (Journal of Cereal Science, 28 (1998)63-70, Evidence for the Presence of a Pentosanase Inhibitor in WheatFlours). Similar to Debyser et al., Rouau and Surget believed that theyhave identified the existence of a thermolabile compound in the solublefraction of wheat flours, which limited the action of an addedpentosanase. Also similarly to Debyser et al., these authors did notisolate an inhibitor and were unable to conclude whether the inhibitoris endogenous or is of microbial origin. Likewise, no chemical data werepresented for this inhibitor.

Thus, a known problem in the art is how to prepare baked goods from adough which does not have adverse handling properties. A more particularproblem is how to provide a dough which is non-sticky—i.e. a dough thatis not so sticky that it causes handling and processing problems.

The present invention seeks to provide a solution to these problems.

SUMMARY ASPECTS OF THE PRESENT INVENTION

Aspects of the present invention are presented in the claims and in thefollowing commentary.

In brief, some aspects of the present invention relate to:

-   -   1. An endogenous endo-β-1,4-xylanase inhibitor—including        nucleotide sequences coding therefor and the amino acid        sequences thereof, as well as variants, homologues, or fragments        thereof.    -   2. Assay methods for determining the effect of the        β-1,4-xylanase inhibitor on different xylanases.    -   3. Assay methods for determining the effect of different        xylanases in dough.    -   4. Assay methods for determining the effect of glucanase(s) on        different doughs containing xylanases.    -   5. Novel xylanases—including nucleotide sequences coding        therefor and the amino acid sequences thereof, as well as        variants, homologues, or fragments thereof.    -   6. Novel uses of xylanases.    -   7. Foodstuffs prepared with xylanases.

Other aspects concerning the amino acid sequence of the presentinvention and/or the nucleotide sequence of the present inventioninclude: a construct comprising or capable of expressing the sequencesof the present invention; a vector comprising or capable of expressingthe sequences of the present invention; a plasmid comprising or capableof expressing the sequences of present invention; a tissue comprising orcapable of expressing the sequences of the present invention; an organcomprising or capable of expressing the sequences of the presentinvention; a transformed host comprising or capable of expressing thesequences of the present invention; a transformed organism comprising orcapable of expressing the sequences of the present invention. Thepresent invention also encompasses methods of expressing the same, suchas expression in a micro-organism; including methods for transferringsame.

The present invention differs from the teachings of WO-A-98/49278because inter alia that PCT patent application contains minimal sequenceinformation regarding the proteinic inhibitor disclosed therein.

Aspects of the present invention are now discussed under appropriatesection headings. For the sake of convenience, generally applicableteachings for the aspects of the present invention may be found in thesections titled “General Definitions” and “General Teachings”. However,the teachings under each section are not necessarily limited to eachparticular section.

GENERAL DEFINITIONS

The term “wheat flour” as used herein is a synonym for the finely-groundmeal of wheat. Preferably, however, the term means flour obtained fromwheat per se and not from another grain. Thus, and unless otherwiseexpressed, references to “wheat flour” as used herein preferably meanreferences to wheat flour per se as well as to wheat flour when presentin a medium, such as a dough.

The term “xylanase” is used in its normal sense—e.g. an enzyme that isinter alia capable of catalysing the depolymerisation of arabinoxylanwhich may be present in wheat (e.g. an enzyme that is inter alia capableof catalysing the solubilisation of WIP and catalysing thedepolymerisation of WSP which may be present in wheat).

An assay for determining endo-β-1,4-xylanase activity is presentedlater. For convenience, this assay is called the “Xylanase Assay”.

The term “nucleotide sequence” in relation to the present inventionincludes genomic DNA, cDNA, recombinant DNA (i.e. DNA prepared by use ofrecombinant DNA techniques), synthetic DNA, and RNA—as well ascombinations thereof.

Preferably, the term “nucleotide sequence” means DNA.

The nucleotide sequences of the present invention may be single ordouble stranded.

The nucleotide sequences of the present invention may include withinthem synthetic or modified nucleotides. A number of different types ofmodification to oligonucleotides are known in the art. These includemethyiphosphonate and phosphorothioate backbones, addition of acridineor polylysine chains at the 3′ and/or 5′ ends of the molecule. For thepurposes of the present invention, it is to be understood that thenucleotide sequences described herein may be modified by any methodavailable in the art. Such modifications may be carried out in toenhance the in vivo activity or life span of nucleotide sequences of thepresent invention.

The terms “variant” or “homologue” with respect to the nucjeotidesequence of the present invention and the amino acid sequence of thepresent invention are synonymous with allelic variations of thesequences.

In particular, the term “homology” as used herein may be equated withthe term “identity”. Here, sequence homology with respect to thenucleotide sequence of the present invention and the amino acid sequenceof the present invention can be determined by a simple “eyeball”comparison (i.e. a strict comparison) of any one or more of thesequences with another sequence to see if that other sequence has atleast 75% identity to the sequence(s). Relative sequence homology (i.e.sequence identity) can also be determined by commercially availablecomputer programs that can calculate % homology between two or moresequences. A typical example of such a computer program is CLUSTAL.

Hence, homology comparisons can be conducted by eye. However, moreusually they are conducted with the aid of readily available sequencecomparison programs. These commercially available computer programs cancalculate % homology between two or more sequences.

% homology may be calculated over contiguous sequences, i.e. onesequence is aligned with the other sequence and each amino acid in onesequence directly compared with the corresponding amino acid in theother sequence, one residue at a time. This is called an “ungapped”alignment. Typically, such ungapped alignments are performed only over arelatively short number of residues (for example less than 50 contiguousamino acids).

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion will cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without penalising unduly the overall homology score. This isachieved by inserting “gaps” in the sequence alignment to try tomaximise local homology.

However, these more complex methods assign “gap penalties” to each gapthat occurs in the alignment so that, for the same number of identicalamino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties will of course produce optimised alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,it is preferred to use the default values when using such software forsequence comparisons. For example when using the GCG Wisconsin Bestfitpackage (see below) the default gap penalty for amino acid sequences is−12 for a gap and −4 for each extension.

Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (University of Wisconsin,U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examplesof other software than can perform sequence comparisons include, but arenot limited to, the BLAST package (see Ausubel et al., 1999 ibid—Chapter18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and theGENEWORKS suite of comparison tools. Both BLAST and FASTA are availablefor offline and online searching (see Ausubel et al., 1999 ibid, pages7-58 to 7-60). However it is preferred to use the GCG Bestfit program.

Although the final % homology can be measured in terms of identity, thealignment process itself is typically not based on an all-or-nothingpair comparison. Instead, a scaled similarity score matrix is generallyused that assigns scores to each pairwise comparison based on chemicalsimilarity or evolutionary distance. An example of such a matrixcommonly used is the BLOSUM62 matrix—the default matrix for the BLASTsuite of programs. GCG Wisconsin programs generally. use either thepublic default values or a custom symbol comparison table if supplied(see user manual for further details). It is preferred to use the publicdefault values for the GCG package, or in the case of other software,the default matrix, such as BLOSUM62.

Once the software has produced an optimal alignment, it is possible tocalculate % homology, preferably % sequence identity. The softwaretypically does this as part of the sequence comparison and generates anumerical result.

Preferably, sequence comparisons are conducted using the simple BLASTsearch algorithm provided at http://www.ncbi.nlm.nih.gov/BLAST using thedefault parameters.

The present invention also encompasses nucleotide sequences that arecomplementary to the sequences presented herein, or any derivative,fragment or derivative thereof. If the sequence is complementary to afragment thereof then that sequence can be used a probe to identifysimilar coding sequences in other organisms etc.

The present invention also encompasses nucleotide sequences that arecapable of hybridising to the sequences presented herein, or anyderivative, fragment or derivative thereof.

The present invention also encompasses nucleotide sequences that arecapable of hybridising to the sequences that are complementary to thesequences presented herein, or any derivative, fragment or derivativethereof.

The term “complementary” also covers nucleotide sequences that canhybridise to the nucleotide sequences of the coding sequence.

The term “variant” also encompasses sequences that are complementary tosequences that are capable of hydridising to the nucleotide sequencespresented herein.

Preferably, the term “variant” encompasses sequences that arecomplementary to sequences that are capable of hydridising understringent conditions (e.g. 65° C. and 0.1×SSC {1×SSC=0.15 M NaCl, 0.015Na₃ citrate pH 7.0}) to the nucleotide sequences presented herein.

The present invention also relates to nucleotide sequences that canhybridise to the nucleotide sequences of the present invention(including complementary sequences of those presented herein).

The present invention also relates to nucleotide sequences that arecomplementary to sequences that can hybridise to the nucleotidesequences of the present invention (including complementary sequences ofthose presented herein).

The term “hybridization” as used herein shall include “the process bywhich a strand of nucleic acid joins with a complementary strand throughbase pairing” (Coombs J (1994) Dictionary of Biotechnology, StocktonPress, New York N.Y.) as well as the process of amplification as carriedout in polymerase chain reaction technologies as described inDieffenbach C W and G S Dveksler (1995, PCR Primer, a Laboratory Manual,Cold Spring Harbor Press, Plainview N.Y.).

Also included within the scope of the present invention arepolynucleotide sequences that are capable of hybridizing to thenucleotide sequences presented herein under conditions of intermediateto maximal stringency. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex, as taught inBerger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methodsin Enzymology, Vol 152, Academic Press, San Diego Calif.), and confer adefined “stringency” as explained below.

Maximum stringency typically occurs at about Tm-5° C. (5° C. below theTm of the probe); high stringency at about 5° C. to 10° C. below Tm;intermediate stringency at about 10° C. to 20° C. below Tm; and lowstringency at about 20° C. to 25° C. below Tm. As will be understood bythose of skill in the art, a maximum stringency hybridization can beused to identify or detect identical polynucleotide sequences while anintermediate (or low) stringency hybridization can be used to identifyor detect similar or related polynucleotide sequences.

In a preferred aspect, the present invention covers nucleotide sequencesthat can hybridise to the nucleotide sequence of the present inventionunder stringent conditions (e.g. 65° C. and 0.1×SSC).

ENDOGENOUS ENDO-β-1,4-XYLANASE INHIBITOR

In one aspect the present invention provides an endogenousendo-β-1,4-xylanase inhibitor that is obtainable from wheat flour.

In our studies, we have found that the inhibitor is a di-peptide, havinga MW of about 40 kDa (as measured by SDS or MS) and that it has a pi ofabout 8 to about 9.5.

In one aspect of the present invention, the inhibitor is in an isolatedform and/or in a substantially pure form. Here, the term “isolated”means that the inhibitor is not in its natural environment.

Sequence analysis to date has revealed the that the inhibitor has atleast one or more of the sequences presented as SEQ ID No. 13, SEQ IDNo. 14, SEQ ID No 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18 and/orSEQ ID No. 19.

Thus, the present invention encompasses an endo-β-1,4-xylanase inhibitorwhich comprises has at least one or more of the sequences presented asSEQ ID No. 13, SEQ ID No.14, SEQ ID No 15, SEQ ID No.16, SEQ ID No.17,SEQ ID No. 18 andior SEQ ID No. 19 or a variant, homologue, or fragmentthereof.

The terms “variant”, “homologue” or “fragment” in relation to theinhibitor of the present invention include any substitution of,variation of, modification of, replacement of, deletion of or additionof one (or more) amino acid from or to the sequence providing theresultant amino acid sequence has xylanase inhibitory action, preferablyhaving at least the same activity as an inhibitor that has at least oneor more of the sequences presented as SEQ ID No.13, SEQ ID No. 14, SEQID No 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18 and/or SEQ ID No.19. In particular, the term “homologue” covers homology with respect tostructure and/or function providing the resultant inhibitor has xylanaseinhibitory action, preferably having at least the same activity of aninhibitor that has at least one or more of the sequences presented asSEQ ID No. 13, SEQ ID No. 14, SEQ ID No 15, SEQ ID No. 16, SEQ ID No.17, SEQ ID No. 18 and/or SEQ ID No. 19. With respect to sequencehomology (i.e. sequence similarity or sequence identity), preferablythere is at least 75%, more preferably at least 80%, more preferably atleast 85%, more preferably at least 90% homology to the sequence shownin the attached sequence listings. More preferably there is at least95%, more preferably at least 98%, homology to the sequence shown in theattached sequence listings.

A putative example of a variant of the inhibitor of the present has atleast one or more of the sequences presented as SEQ. ID No. 1 and SEQ.ID No. 2.

The inhibitor aspect of the present invention is advantageous for anumber of reasons.

By way of example, by now knowing the chemical identity of an endogenousendo-β-1,4-xylanase inhibitor workers can now determine the quantity ofthe inhibitor in, for example, a wheat flour. For convenience, we shallcall this method the “Inhibitor Amount Determination Method”.

The Inhibitor Amount Determination Method would enable workers to selectone or more appropriate xylanases for addition to the wheat flour and/orselect appropriate amounts of one or more xylanases for addition to thewheat flour.

Thus, the present invention provides a method comprising: (a)determining the amount or type of inhibitor in a wheat flour; (b)selecting a suitable xylanase for addition to the wheat flour and/orselecting a suitable amount of a xylanase for addition to the wheatflour; and (c) adding the suitable xylanase and/or suitable amount ofthe xylanase to the wheat flour.

The present invention also provides a method comprising: (a) determiningthe amount or type of inhibitor in a wheat flour; (b) selecting asuitable xylanase inhibitor for addition to the wheat flour and/orselecting a suitable amount of a xylanase inhibitor for addition to thewheat flour; and (c) adding the suitable xylanase inhibitor and/orsuitable amount of the xylanase inhibitor to the wheat flour.

The present invention also provides a method comprising: (a) determiningthe amount or type of inhibitor in a wheat flour; (b) selecting asuitable xylanase and a suitable xylanase inhibitor for addition to thewheat flour and/or selecting a suitable amount of a xylanase inhibitorfor addition to the wheat flour; and (c) adding the suitable xylanaseand the suitable xylanase inhibitor and/or suitable amount of thexylanase inhibitor to the wheat flour.

Detection of the amount of inhibitor can be determined by standardchemical techniques, such as by analysis of solid state NMR spectra. Theamount of inhibitor may even be determined by use of xylanase enzymesthat are known to be detrimentally affected by the inhibitor. In thislast aspect, it would be possible to take a sample of the wheat flourand add it to a known quantity of such a xyla.nase. At a certain timepoint the activity of the xylanase can be determined, which resultantactivity can then be correlated to an amount of inhibitor in the wheatflour.

Thus, the present invention also encompasses the use of the combinationof a xylanase and the inhibitor as a means to calibrating and/ordetermining the quantity of inhibitor in a wheat flour sample.

Antibodies to the inhibitor can be used to screen wheat flour samplesfor the presence of the inhibitor of the present invention. Theantibodies may even be used to isolate amounts of the inhibitor from awheat flour sample.

ASSAY METHODS FOR DETERMINING THE EFFECT OF THE β-1,4-XYLANASE INHIBITORON DIFFERENT XYLANASES

There is an additional important use of the inhibitor of the presentinvention.

In this respect, the inhibitor could be used in an assay/screen toidentify xylanases that are affected by the inhibitor.

By way of example, in some circumstances, it may be desirable to screenfor a xylanase that has a low resistance—i.e. are not that resistant—tothe inhibitor.

In one aspect, the inhibitor can be used in an assay/screen to identifyxylanases that have a fair (medium) resistance—i.e. are reasonablyresistant—to the inhibitor.

In one aspect, the inhibitor can be used in an assay/screen to identifyxylanases that have a high resistance to the inhibitor.

A suitable Protocol for determining the degree of inhibition by theinhibitor is presented later on. For convenience, we shall call thisProtocol “Inhibitor Assay Protocol”.

Thus, the present invention provides a method for determining the degreeof resistance of a xylanase to a xylanase inhibitor, wherein the methodcomprises: (a) contacting a xylanase of interest with the inhibitor; and(b) determining whether the inhibitor inhibits the activity of thexylanase of interest. For convenience, we shall call this method the“Inhibitor Assay Method”.

Here, the term “resistant” means that the activity of the xylanase isnot totally inhibited by the inhibitor. In other words, the inhibitorcan be used in an assay/screen to identify xylanases that are notdetrimentally affected by the inhibitor.

Thus, the term “degree of resistance” in relation to the xylanasevis-a-vis the xylanase inhibitor is synonymous with the degree ofnon-inhibition of the activity of a xylanase by the xylanase inhibitor.Thus, a xylanase that has a high degree of resistance to the xylanaseinhibitor is akin to a high degree of non-inhibition of a xylanase bythe xylanase inhibitor.

The present invention also encompasses a process comprising the steps of(a) performing the Inhibitor Assay Method; (b) identifying one or morexylanases having a high (or medium or low) degree of resistance to theinhibitor; (c) preparing a quantity of those one or more identifiedxylanases.

Suitable identified xylanases can then be used to prepare a foodstuff,in particular a dough to make a bakery product.

In addition, by identifying a xylanase that is resistant to some extentto the inhibitor (i.e. a xylanase that is not inhibited as much as otherxylanases), it is possible to add less of that identified xylanase to amedium for subsequent utilisation thereof. End uses for the xylanasescan include any one or more of the preparation of foodstuffs, proteinand starch production, paper production and pulp processing etc.

Thus, the present invention also encompasses a process comprising thesteps of: (a) performing the Inhibitor Assay Method; (b) identifying oneor more xylanases having a high (or medium or low) degree of resistanceto the inhibitor; and (c) preparing a dough comprising the one or moreidentified xylanases.

In the course of the experiments relating to the present invention, wesurprisingly found that bacterial xylanases were able to be resistant tothe inhibitor, in the sense that their activity was not compeletlyabolished. In some cases, the xylanases exhibited very favourableresistance to the inhibitor.

ASSAY METHODS FOR DETERMINING THE EFFECT OF DIFFERENT XYLANASES INDOUGHS

When some bacterial xylanases that had been identified as being suitableby the Inhibitor Assay Method were present in a dough mixture, wesurprisingly found that the dough mixture was not as sticky as a doughmixture comprising a fungal xylanase. These results were completelyunexpected in view of the teachings of the prior art.

Thus, the present invention provides a further assay method foridentifying a bacterial xylanase or mutant thereof suitable for use inthe preparation of a baked foodstuff. The method comprises (a)incorporating a bacterial xylanase of interest in a dough mixture; and(b) determining the stickiness of the resultant dough mixture; such thatthe bacterial xylanase or mutant thereof is suitable for use in thepreparation of a baked foodstuff if the resultant dough mixture has astickiness that is less than a similar dough mixture comprising a fungalxylanase. For convenience, we shall call this method the “StickinessAssay Method”.

Thus, the present invention also provides a process comprising the stepsof: (a) performing the Stickiness Assay Method; (b) identifying one ormore xylanases suitable for use in the preparation of a baked foodstuff;(c) preparinga quantity of those one or more identified xylanases.

A suitable Protocol for determining the stickiness of a dough ispresented later on. For convenience, we shall call this Protocol the“Stickiness Protocol”. In accordance with the present invention a doughcomprising a xylanase according to the present invention that is lesssticky than a dough comprising a fungal xylanase may be called, onoccassion, a “non-sticky dough”.

If a bacterial xylanase shows favourable properties—in that it does notproduce a dough that is as sticky as a dough comprising a fungalxylanase—then that xylanase may be used to prepare a foodstuff, such asa dough for preparing a bakery product.

Thus, the present invention also provides a process comprising the stepsof: (a) performing the Stickiness Assay Method; (b) identifying one ormore xylanases suitable for use in the preparation of a baked foodstuff;and (c) preparing a dough comprising the one or more identifiedxylanases.

ASSAY METHODS FOR DETERMINING THE EFFECT OF GLUCANASE(S) ON DOUGHPROPERTIES FOR DOUGHS THAT MAY COMPRISE XYLANASES

In the course of the experiments relating to the present invention, wealso found that the presence of glucanase enzymes in certain amountscould have a detrimental effect on the xylanases.

Thus, in one aspect, it is advantageous not to have detrimental levelsof glucanase enzymes in the xylanase preparation—such as the medium usedto prepare or extract the xylanase enzymes. In addition, for someaspects, it is advantageous not to have detrimental levels of glucanaseenzymes in a medium that is to be used to prepare a foodstuff whichmedium will contain the xylanase. Here, the term “detrimental level”means an amount of glucanase is present such that the benefits from thexylanase are masked by the adverse effect of the glucanase enzymes.

Thus, the present invention provides a further assay method foridentifying a xylanase composition (such as a xylanase preparation) or amedium in which a xylanase is to be prepared or a medium to which axylanase is to be added that is to be suitable for use in thepreparation of a baked foodstuff, the method comprising (a) providing acomposition containing the xylanase of interest or a medium in which thexylanase is to be prepared or a medium to which the xylanase is to beadded; and (b) determining the presence of active glucanase enzyme(s) inthe composition or medium; such that if there is at most a low level ofactive glucanase enzyme(s) in the composition or medium then thatcomposition or medium is suitable for the preparation of a bakedfoodstuff. For convenience, we shall call this method the “GlucanaseAssay Method”.

The present invention also provides a process comprising the steps of:(a) performing the Glucanase Assay Method; (b) identifying one or morecompositions or mediums suitable for use in the preparation of a bakedfoodstuff; (c) preparing a quantity of those one or more identifiedcompositions or mediums.

A suitable Protocol for determining the activity of glucanases ispresented later on. For convenience, we shall call this Protocol the“Glucanase Protocol”.

If the composition or medium shows favourable properties—in the sensethat the beneficial effects associated with the xylanase are notcompletely masked by the presence of detrimental amounts of glucanaseenzymes—then that composition or medium may be used to prepare afoodstuff, preferably dough that is used to make a bakery product.

Thus, the present invention also encompasses a process comprising thesteps of: (a) performing the Glucanase Assay Method; (b) identifying oneor more identified compositions or mediums suitable for use in thepreparation of a baked foodstuff; and (c) preparing a dough comprisingthe one or more identified identified compositions or mediums.

Thus, the present invention covers a xylanase preparation, wherein thexylanase preparation is substantially free of glucanase enzyme(s).

In this respect, the xylanase preparation can be prepared from aninitial preparation from which at least substantially all of theglucanase enzyme(s) that may be present is(are) removed or even whereinthe activity of the glucanase enzyme(s) is suppressed or eliminated.Techniques for achieving this could include using antibodies thatrecognise and bind to the glucanase enzyme(s) and in doing so inactivatethe activity of the glucanase enzyme(s). Alternatively, glucanaseenzyme(s) specific antibodies could be bound to a support such thatpassage of the initial preparation past the bound antibodies wouldresult in the glucanase enzyme(s) being removed from it thereby forminga xylanase preparation being substantially free of glucanase enzyme(s).In an alternative embodiment, or even in an additional embodiment, thexylanase preparation can be prepared from a host organism that hasminimal or no glucanase enzyme activity. In this aspect, the activity ofthe glucanase enzymes that are present in the host organism may beinactivated. In an alternative aspect, the expression of the glucanasegenes can be silenced andlor knocked-out. Techniques for achieving thiscould include using antisense sequences to the glucanase codingsequences. In a further embodiment, a host organism is used that has noor at most minimal expression of glucanase enzymes.

K_(i) ASSAY

In some cases, measurement of the K_(i) value of a xylanase (which wecall here a “K_(i) assay”) may be useful. In this respect, we. havefound that in some cases the K_(i) value is sometimes indicative of thesuitability of the xylanase for certain application(s). Knowledge of theK_(i) value could be useful on its own.

COMBINATION ASSAYS

The present invention also encompasses suitable combinations of theassays of the present invention.

In this respect, the present invention includes a combination methodcomprising two or more of the following steps: a step comprising theInhibitor Amount Determination Method, a step comprising the InhibitorAssay Method, a step comprising the Stickiness Assay Method; a stepcomprising the Glucanase Assay Method; and a step comprising the K_(i)assay. In the combination method, the steps can occur in any order andneed not necessarily occur simulataneously or consecutively.

NOVEL XYLANASES

As indicated above, the present invention provides a suitable assay foridentifying xylanases that can be used in the preparation of foodstuffs,in particular doughs for use in the preparation of bakery products.

In this respect, we have identified three new xylanases that aresuitable for the preparation of foodstuffs, in particular doughs for usein the preparation of bakery products.

Thus, the present invention also includes an amino acid sequencecomprising any one of the amino acid sequences presented as SEQ ID No.7, SEQ ID No. 9 or SEQ ID No. 11, or a variant, homologue or fragmentthereof.

The terms “variant”, “homologue” or “fragment” in relation to thexylanase of the present invention include any substitution of, variationof, modification of, replacement of, deletion of or addition of one (ormore) amino acid from or to the sequence providing the resultant aminoacid sequence has xylanase activity, preferably having at least the sameactivity comprising any one of the amino acid sequences presented as SEQID No. 7, SEQ ID No. 9 or SEQ ID No. 11. In particular, the term“homologue” covers homology with respect to structure and/or functionproviding the resultant protein has xylanase activity, preferably atleast the same activity of any one of the amino acid sequences presentedas SEQ ID No. 7, SEQ ID No. 9 or SEQ ID No. 11. With respect to sequencehomology (i.e. sequence similarity or sequence identity), preferablythere is at least 75%, more preferably at least 85%, more preferably atleast 90% homology to the sequence shown in the attached sequencelistings. More preferably there is at least 95%, more preferably atleast 98%, homology to the sequence shown in the attached sequencelistings.

Preferably, the xylanase comprises the sequence presented as SEQ ID No.7 or SEQ ID No. 11, or a variant, homologue or fragment thereof.

The present invention also encompasses a nucleotide sequence encodingthe amino acid sequence of the present invention.

Preferably, the nucleotide sequence of the present invention is selectedfrom:

-   -   (a) a nucteotide sequence comprising any one of the nucleotide        sequences presented as SEQ ID No. 8, SEQ ID No. 10 or SEQ ID No.        12, or a variant, homologue or fragment thereof;    -   (b) any one of the nucleotide sequences presented as SEQ ID No.        8, SEQ ID No. 10 or SEQ ID No. 12, or the complement thereof;    -   (c) a nucleotide sequence capable of hybridising any one of the        nucleotide sequences presented as SEQ ID No. 8, SEQ ID No. 10 or        SEQ ID No. 12, or a fragment thereof;    -   (d) a nucleotide sequence capable of hybridising to the        complement any one of the nucleotide sequences presented as SEQ        ID No. 8, SEQ ID No. 10 or SEQ ID No. 12, or a fragment thereof;        and    -   (e) a nucleotide sequence which is degenerate as a result of the        genetic code to the nucleotides defined in (a), (b), (c) or (d).

The terms “variant”, “homologue” or“fragment” in relation to thenucleotide sequence of the present invention include any substitutionof, variation of, modification of, replacement of, deletion of oraddition of one (or more) nucleic acid from or to the sequence providingthe resultant nucleotide sequence codes for an amino acid sequence hasxylanase activity, preferably having at least the same activitycomprising any one of the amino acid sequences presented as SEQ ID No.7, SEQ ID No. 9 or SEQ ID No. 11. In particular, the term “homologue”covers homology with respect to structure and/or function providing theresultant expressed protein has xylanase activity, preferably at leastthe same activity of any one of the amino acid sequences presented asSEQ ID No. 7, SEQ ID No. 9 or SEQ ID No. 11. With respect to sequencehomology (i.e. sequence similarity or sequence identity), preferablythere is at least 75%, more preferably at least 85%, more preferably atleast 90% homology to the sequence shown as SEQ ID No. 8, SEQ ID No. 10or SEQ ID No. 12 in the attached sequence listings. More preferablythere is at least 95%, more preferably at least 98%, homology to thesequence shown in the attached sequence listings.

Preferably, the nucleotide sequence of the present invention comprisesthe sequence presented as SEQ ID No. 8 or SEQ ID No. 12, or a variant,homologue or fragment thereof.

NOVEL USES OF XYLANASES

As indicated above, the present invention also provides a suitable assayfor identifying xylanases that can be used in the preparation ofnon-sticky doughs (as defined herein) for use in the preparation ofbakery products.

In this respect, we have identified certain xylanases, both known andnew bacterial xylanases, that are suitable for the preparation offoodstuffs, in particular doughs for use in the preparation of bakeryproducts.

Thus, the present invention covers a non-sticky dough (as hereindefined) which dough comprises a xylanase identifiable by the assay ofthe present invention. Preferably, the xylanase has an amino acidsequence presented as any one of SEQ ID No.s 3, 5, 7, 9, 11, or avariant, derivative or homologue thereof. More preferably, the xylanasehas an amino acid sequence presented as any one of SEQ ID No.s 5, 7, 9,11, or a variant, derivative or homologue thereof.

In contrast to the prior art systems, the present invention provides forthe possibility of the addition of xylanase directly to flour prior toproduction of the dough. Thus, a single batch a flourixylanase mixturemay be delivered to the dough producer. Moreover, the dough producerdoes not require dosing equipment to be able to obtain a readilyhandable dough.

FOODSTUFFS PREPARED WITH XYLANASES

The present invention provides a means of identifying suitable xylanasesfor use in the manufacture of a foodstuff. Typical foodstuffs, whichalso include animal feed, include dairy products, meat products, poultryproducts, fish products and bakery products.

Preferably, the foodstuff is a bakery product. Typical bakery (baked)products incorporated within the scope of the present invention includebread—such as loaves, rolls, buns, piza bases etc.—pretzels, tortillas,cakes, cookies, biscuits, crackers etc.

GENERAL TEACHINGS

In the following commentary references to “nucleotide sequence of thepresent invention” and “amino acid sequence of the present invention”refer respectively to any one or more of the nucteotide sequences.presented herein and to any one or more of the amino acid sequencespresent herein.

Amino Acid Sequence/Polypeptide Sequence

The term “amino acid sequence of the present invention” is synonymouswith the phrase “polypeptide sequence of the present invention”. Here,the amino acid sequence may be that for the xylanase or the xylanaseinhibitor.

Polypeptides of the present invention also include fragments of thepresented amino acid sequence and variants thereof. Suitable fragmentswill be at least 5, e.g. at least 10, 12, 15 or 20 amino acids in size.

Polypeptides of the present invention may also be modified to containone or more (e.g. at least 2, 3, 5, or 10) substitutions, deletions orinsertions, including conserved substitutions.

Conserved substitutions may be made according to the following tablewhich indicates conservative substitutions, where amino acids on thesame block in the second column and preferably in the same line in thethird column may be substituted for each. other: ALIPHATIC Non-polar G AP I L V Polar - uncharged C S T M N Q Polar - charged D E K R AROMATIC HF W Y OTHER N Q D E

Polypeptides of the present invention may be in a substantially isolatedform. It will be understood that the polypeptide may be mixed withcarriers or diluents which will not interfere with the intended purposeof the polypeptide and still be regarded as substantially isolated. Apolypeptide of the present invention may also be in a substantiallypurified form, in which case it will generally comprise the polypeptidein a preparation in which more than 90%, e.g. 95%, 98% or 99% of thepolypeptide in the preparation is a polypeptide of the presentinvention. Polypeptides of the present invention may be modified forexample by the addition of histidine residues to assist theirpurification or by the addition of a signal sequence to promote theirsecretion from a cell as discussed below.

Polypeptides of the present invention may be produced by synthetic means(e.g. as described by Geysen et al., 1996) or recombinantly, asdescribed below.

The use of suitable host cells—such as yeast, fungal and plant hostcells—may provide for such post-translational modifications (e.g.myristolation, glycosylation, truncation, lapidatioh and tyrosine,serine or threonine phosphorylation) as may be needed to confer optimalbiological activity on recombinant expression products of the presentinvention.

Nucleotide Sequence/Polynucleotide Sequence

The term “nucleotide sequence of the present invention” is synonymouswith the phrase “polynucleotide sequence of the present invention”.

Polynucleotides of the present invention include nucleotide acidsequences encoding the polypeptides of the present invention. It willappreciated that a range of different polynucleotides encode a givenamino acid sequence as a consequence of the degeneracy of the geneticcode.

By knowledge of the amino acid sequences set out herein it is possibleto devise partial and full-length nucleic acid sequences such as cDNAand/or genomic clones that encode the polypeptides of the presentinvention. For example, polynucleotides of the present invention may beobtained using degenerate PCR which will use primers designed to targetsequences encoding the amino acid sequences presented herein. Theprimers will typically contain multiple degenerate positions. However,to minimise degeneracy, sequences will be chosen that encode regions ofthe amino acid sequences presented herein containing amino acids such asmethionine which are coded for by only one triplet. In addition,sequences will be chosen to take into account codon usage in theorganism whose nucleic acid is used as the template DNA for the PCRprocedure. PCR will be used at stringency conditions lower than thoseused for cloning sequences with single sequence (non-denegerate) primersagainst known sequences.

Nucleic acid sequences obtained by PCR that encode polypeptide fragmentsof the present invention may then be used to obtain larger sequencesusing hybridization library screening techniques. For example a PCRclone may be labelled with radioactive atoms and used to screen a cDNAor genomic library from other species, preferably other plant species orfungal species. Hybridization conditions will typically be conditions ofmedium to high stringency (for example 0.03M sodium chloride and 0.03Msodium citrate at from about 50° C. to about 60° C.).

Degenerate nucleic acid probes encoding all or part of the amino acidsequence may also be used to probe cDNA and/or genomic libraries fromother species, preferably other plant species or fungal species.However, it is preferred to carry out PCR techniques initially to obtaina single sequence for use in further screening procedures.

Polynucleotide sequences of the present invention obtained using thetechniques described above may be used to obtain further homologoussequences and variants using the techniques described above. They mayalso be modified for use in expressing the polypeptides of the presentinvention in a variety of host cells systems, for example to optimisecodon preferences for a particular host cell in which the polynucleotidesequences are being expressed. Other sequence changes may be desired inorder to introduce restriction enzyme recognition sites, or to alter theproperty or function of the polypeptides encoded by the polynucleotides.

Polynucleotides of the present invention may be used to produce aprimer, e.g. a PCR primer, a primer for an alternative amplificationreaction, a probe e.g. labelled with a revealing label by conventionalmeans using radioactive or non-radioactive labels, or thepolynucleotides may be cloned into vectors. Such primers, probes andother fragments will be at least 15, preferably at least 20, for exampleat least 25, 30 or 40 nucleotides in length, and are also encompassed bythe term polynucleotides of the present invention as used herein.

Polynucleotides or primers of the present invention may carry arevealing label. Suitable labels include radioisotopes such as ³²P or³⁵S, enzyme labels, or other protein labels such as biotin. Such labelsmay be added to polynucleotides or primers of the present invention andmay be detected using by techniques known per se.

Polynucleotides such as a DNA polynucleotide and primers according tothe present invention may be produced recombinantly, synthetically, orby any means available to those of skill in the art. They may also becloned by standard techniques.

In general, primers will be produced by synthetic means, involving astep wise manufacture of the desired nucleic acid sequence onenuclectide at a time. Techniques for accomplishing this using automatedtechniques are readily available in the art.

Longer polynucleotides will generally be produced using recombinantmeans, for example using a PCR (polymerase chain reaction) cloningtechniques. This will involve making a pair of primers (e.g. of about15-30 nucleotides) to a region of the endo-β-1,4-xylanase inhibitor genewhich it is desired to clone, bringing the primers into contact withmRNA or cDNA obtained from a fungal, plant or prokaryotic cell,performing a polymerase chain reaction under conditions which bringabout amplification of the desired region, isolating the amplifiedfragment (e.g. by purifying the reaction mixture on an agarose gel) andrecovering the amplified DNA. The primers may be designed to containsuitable restriction enzyme recognition sites so that the amplified DNAcan be cloned into a suitable cloning vector.

Regulatory Sequences

Preferably, the polynucleotide of the present invention is operablylinked to a regulatory sequence which is capable of providing for theexpression of the coding sequence, such as by the chosen host cell. Byway of example, the present invention covers a vector comprising thepolynucleotide of the present invention operably linked to such aregulatory sequence, i.e. the vector is an expression vector.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A regulatory sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under condition compatible with the controlsequences.

The term “regulatory sequences” includes promoters and enhancers andother expression regulation signals.

The term “promoter” is used in the normal sense of the art, e.g. an RNApolymerase binding site.

Enhanced expression of the polynucleotide encoding the polypeptide ofthe present invention may also be achieved by the selection ofheterologous regulatory regions, e.g. promoter, secretion leader andterminator regions, which serve to increase expression and, if desired,secretion levels of the protein of interest from the chosen expressionhost and/or to provide for the inducible control of the expression ofthe polypeptide of the present invention

Preferably, the nucleotide sequence of the present invention may beoperably linked to at least a promoter.

Aside from the promoter native to the gene encoding the polypeptide ofthe present invention, other promoters may be used to direct expressionof the polypeptide of the present invention. The promoter may beselected for its efficiency in directing the expression of thepolypeptide of the present invention in the desired expression host.

In another embodiment, a constitutive promoter may be selected to directthe expression of the desired polypeptide of the present invention. Suchan expression construct may provide additional advantages since itcircumvents the need to culture the expression hosts on a mediumcontaining an inducing substrate.

Examples of strong constitutive and/or inducible promoters which arepreferred for use in fungal expression hosts are those which areobtainable from the fungal genes for xylanase (xlnA), phytase,ATP-synthetase, subunit 9 (oliC), triose phosphate isomerase (tpi),alcohol dehydrogenase (AdhA), α-amylase (amy), amyloglucosidase (AG—fromthe glaA gene), acetamidase (amdS) and glyceraldehyde-3-phosphatedehydrogenase (gpd) promoters.

Examples of strong yeast promoters are those obtainable from the genesfor alcohol dehydrogenase, lactase, 3-phosphoglycerate kinase andtriosephosphate isomerase.

Examples of strong bacterial promoters are the α-amylase and SP02promoters as well as promoters from extracellular protease genes.

Hybrid promoters may also be used to improve inducible regulation of theexpression construct.

The promoter can additionally include features to ensure or to increaseexpression in a. suitable host. For example, the features can beconserved regions such as a Pribnow Box or a TATA box. The promoter mayeven contain other sequences to affect (such as to maintain, enhance,decrease) the levels of expression of the nucleotide sequence of thepresent invention. For example, suitable other sequences include theSh1-intron or an ADH intron. Other sequences include inducibleelements—such as temperature, chemical, light or stress inducibleelements. Also, suitable elements to enhance transcription ortranslation may be present. An example of the latter element is the TMV5′ signal sequence (see Sleat Gene 217 [1987] 217-225; and Dawson PlantMol. Biol. 23 [1993] 97).

Secretion

Often, it is desirable for the polypeptide of the present invention tobe secreted from the expression host into the culture medium from wherethe polypeptide of the present invention may be more easily recovered.According to the present invention, the secretion leader sequence may beselected on the basis of the desired expression host. Hybrid signalsequences may also be used with the context of the present invention.

Typical examples of heterologous secretion leader sequences are thoseoriginating from the fungal amyloglucosidase (AG) gene (glaA—both 18 and24 amino acid versions e.g. from Aspergillus), the a-factor gene (yeastse.g. Saccharomyces and Kluyveromyces) or the α-amylase gene (Bacillus).

Constructs

The term “construct”—which is synonymous with terms such as “conjugate”,“cassette” and “hybrid”—includes the nucleotide sequence according tothe present invention directly or indirectly attached to a promoter. Anexample of an indirect attachment is the provision of a suitable spacergroup such as an intron sequence, such as the Sh1-intron or the ADHintron, intermediate the promoter and the nucleotide sequence of thepresent invention. The same is true for the term “fused” in relation tothe present invention which includes direct or indirect attachment Ineach case, the terms do not cover the natural combination of thenucleotide sequence coding for the protein ordinarily associated withthe wild type gene promoter and when they are both in their naturalenvironment.

The construct may even contain or express a marker which allows for theselection of the genetic construct in, for example, a bacterium,preferably of the genus Bacillus, such as Bacillus subtilis, or plants,such as potatoes, sugar beet etc., into which it has been transferred.Various markers exist which may be used, such as for example thoseencoding mannose-6-phosphate isomerase (especially for plants) or thosemarkers that provide for antibiotic resistance—e.g. resistance to G418,hygromycin, bleomycin, kanamycin and gentamycin.

Preferably the construct of the present invention comprises at least thenucleotide sequence of the present invention operably linked to apromoter.

Vectors

The term “vector” includes expression vectors and transformation vectorsand shuttle vectors.

The term “expression vector” means a construct capable of in vivo or invitro expression.

The term “transformation vector” means a construct capable of beingtransferred from one entity to another entity—which may be of thespecies or may be of a different species. If the construct is capable ofbeing transferred from one species to another—such as from an E. coliplasmid to a bacterium, preferably of the genus Bacillus, then thetransformation vector is sometimes called a “shuttle vector”. It mayeven be a construct capable of being transferred from an E. coli plasmidto an Agrobacterium to a plant.

The vectors of the present invention may be transformed into a suitablehost cell as described below to provide for expression of a polypeptideof the present invention. Thus, in a further aspect the inventionprovides a process for preparing polypeptides according to the presentinvention which comprises cultivating a host cell transformed ortransfected with an expression vector as described above underconditions to provide for expression by the vector of a coding sequenceencoding the polypeptides, and recovering the expressed polypeptides.

The vectors may be for example, plasmid, virus or phage vectors providedwith an origin of replication, optionally a promoter for the expressionof the said polynucleotide and optionally a regulator of the promoter.

The vectors of the present invention may contain one or more selectablemarker genes. The most suitable selection systems for industrialmicro-organisms are those formed by the group of selection markers whichdo not require a mutation in the host organism. Examples of fungalselection markers are the genes for acetamidase (amdS), ATP synthetase,subunit 9 (oliC), orotidine-5′-phosphate-decarboxylase (pvrA),phleomycin and benomyl resistance (benA). Examples of non-fungalselection markers are the bacterial G418 resistance gene (this may alsobe used in yeast, but not in fungi), the ampicillin resistance gene (E.coli), the neomycin resistance gene (Bacillus) and the E. coli uidAgene, coding for β-glucuronidase (GUS).

Vectors may be used in vitro, for example for the production of RNA orused to transfect or transform a host cell.

Thus, polynucleotides of the present invention can be incorporated intoa recombinant vector (typically a replicable vector), for example acloning or expression vector. The vector may be used to replicate thenucleic acid in a compatible host cell. Thus in a further embodiment,the invention provides a method of making polynucleotides of the presentinvention by introducing a polynucleotide of the present invention intoa replicable vector, introducing the vector into a compatible host cell,and growing the host cell under conditions which bring about replicationof the vector. The vector may be recovered from the host cell. Suitablehost cells are described below in connection with expression vectors.

Tissue

The term “tissue” as used herein includes tissue per se and organ.

Host Cells

The term “host cell”—in relation to the present invention includes anycell that could comprise the nucleotide sequence coding for therecombinant protein according to the present invention and/or productsobtained therefrom, wherein a promoter can allow expression of thenucleotide sequence according to the present invention when present inthe host cell.

Thus, a further embodiment of the present invention provides host cellstransformed or transfected with a polynucleotide of the presentinvention. Preferably said polynucleotide is carried in a vector for thereplication and expression of said polynucleotides. The cells will bechosen to be compatible with the said vector and may for example beprokaryotic (for example bacterial), fungal, yeast or plant cells.

The gram-negative bacterium E. coli is widely used as a host forheterologous gene expression. However, large amounts of heterologousprotein tend to accumulate inside the cell. Subsequent purification ofthe desired protein from the bulk of E. coli intracellular proteins cansometimes be difficult.

In contrast to E. coli, bacteria from the genus Bacillus are verysuitable as heterologous hosts because of their capability to secreteproteins into the culture medium. Other bacteria suitable as hosts arethose from the genera Streptomyces and Pseudomonas.

Depending on the nature of the polynucleotide encoding the polypeptideof the present invention, and/or the desirability for further processingof the expressed protein, eukaryotic hosts such as yeasts or fungi maybe preferred. In general, yeast cells are preferred over fungal cellsbecause they are easier to manipulate. However, some proteins are eitherpoorly secreted from the yeast cell, or in some cases are not processedproperly (e.g. hyperglycosylation in yeast)., In these instances, afungal host organism should be selected.

Examples of preferred expression hosts within the scope of the presentinvention are fungi such as Aspergillus species (such as those describedin EP-A-0184438 and EP-A-0284603) and Trichoderma species; bacteria suchas Bacillus species (such as those described in EP-A-0134048 andEP-A-0253455), Streptomyces species and Pseudomonas species; and yeastssuch as Kluyveromyces species (such as those described in EP-A-0096430and EP-A-0301670) and Saccharomyces species.

Typical expression hosts may be selected from Aspergillus niger,Aspergillus niger var. tubigenis, Aspergillus niger var. awamori,Aspergillus aculeatis, Aspergillus nidulans, Aspergillus orvzae,Trichoderma reesei, Bacillus subtilis, Bacillus licheniformis, Bacillusamyloliquefaciens, Kluyveromyces lactis and Saccharomyces cerevisiae.

Organism

The term “organism” in relation to the present invention includes anyorganism that could comprise the nucleotide sequence coding for therecombinant protein according to the present invention and/or productsobtained therefrom, wherein a promoter can allow expression of thenucleotide sequence according to the present invention when present inthe organism. For the xylanase inhibitor aspect of the presentinvention, preferable organisms may include a fugus, yeast or a plant.For the xylanase aspect of the present invention, a preferabe organismmay be a bacterium, preferably of the genus Bacillus, more preferablyBacillus subtilis.

The term “transgenic organism” in relation to the present inventionincludes any organism that comprises the nucleotide sequence coding forthe protein according to the present invention and/or products obtainedtherefrom, wherein the promoter can allow expression of the nucleotidesequence according to the present invention within the organism.Preferably the nucleotide sequence is incorporated in the genome of theorganism.

The term “transgenic organism” does not cover the native nucleotidecoding sequence according to the present invention in its naturalenvironment when it is under the control of its native promoter which isalso in its natural environment. In addition, the present invention doesnot cover the native protein according to the present invention when itis in its natural environment and when it has been expressed by itsnative nucleotide coding sequence which is also in its naturalenvironment and when that nucleotide sequence is under the control ofits native promoter which is also in its natural environment.

Therefore, the transgenic organism of the present invention includes anorganism comprising any one of, or combinations of, the nucleotidesequence coding for the amino acid sequence according to the presentinvention, constructs according to the present invention (includingcombinations thereof), vectors according to the present invention,plasmids according to the present invention, cells according to thepresent invention, tissues according to the present invention or theproducts thereof. The transformed cell or organism could prepareacceptable quantities of the desired compound which would be easilyretrievable from, the cell or organism.

Transformation of Host Cells/Host Organisms

As indicated earlier, the host organism can be a prokaryotic or aeukaryotic organism. Examples of suitable prokaryotic hosts include E.coli and Bacillus subtilis. Teachings on the transformation ofprokaryotic hosts is well documented in the art, for example seeSambrook et al (Molecular Cloning: A Laboratory Manual, 2nd edition,1989, Cold Spring Harbor Laboratory Press) and Ausubel et al., CurrentProtocols in Molecular Biology (1995), John Wiley & Sons, lnc.

If a prokaryotic host is used then the nucleotide sequence may need tobe suitably modified before transformation—such as by removal ofintrons.

As mentioned above, a preferred host organism is of the genus Bacillus,such as Bacillus subtilis.

In another embodiment the transgenic organism can be a yeast. In thisregard, yeast have also been widely used as a vehicle for heterologousgene expression. The species Saccharomyces cerevisiae has a long historyof industrial use, including its use for heterologous gene expression.Expression of heterologous genes in Saccharomyces cerevisiae has beenreviewed by Goodey et al (1987, Yeast Biotechnology, D R Berry et al,eds, pp 401-429, Allen and Unwin, London) and by King et al (1989,Molecular and Cell Biology of Yeasts, E F Walton and G T Yarronton, eds,pp 107-133, Blackie, Glasgow).

For several reasons Saccharomyces cerevisiae is well suited forheterologous gene expression. First, it is non-pathogenic to humans andit is incapable of producing certain endotoxins. Second, it has a longhistory of safe use following centuries of commercial exploitation forvarious purposes. This has led to wide public acceptability. Third, theextensive commercial use and research devoted to the organism hasresulted in a wealth of knowledge about the genetics and physiology aswell as large-scale fermentation characteristics of Saccharomycescerevisiae.

A review of the principles of heterologous gene expression inSaccharomyces cerevisiae and secretion of gene products is given by EHinchcliffe E Kenny (1993, “Yeast as a vehicle for the expression ofheterologous genes”, Yeasts, Vol 5, Anthony H Rose and J StuartHarrison, eds, 2nd edition, Academic Press Ltd.).

Several types of yeast vectors are available, including integrativevectors, which require recombination with the host genome for theirmaintenance, and autonomously replicating plasmid vectors.

In order to prepare the transgenic Saccharomyces, expression constructsare prepared by inserting the nucleotide sequence of the presentinvention into a construct designed for expression in yeast. Severaltypes of constructs used for heterologous expression have beendeveloped. The constructs contain a promoter active in yeast fused tothe nucleotide sequence of the present invention, usually a promoter ofyeast origin, such as the GAL1 promoter, is used. Usually a signalsequence of yeast origin, such as the sequence encoding the SUC2 signalpeptide, is used. A terminator active in yeast ends the expressionsystem.

For the transformation of yeast several transformation protocols havebeen developed. For example, a transgenic Saccharomyces according to thepresent invention can be prepared by following the teachings of Hinnenet al (1978, Proceedings of the National Academy of Sciences of the USA75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and Ito, H et al(1983, J Bacteriology 153, 163-168).

The transformed yeast cells are selected using various selectivemarkers. Among the markers used for transformation are a number ofauxotrophic markers such as LEU2, HIS4 and TRP1, and dominant antibioticresistance markers such as aminoglycoside antibiotic markers, eg G418.

Another host organism is a plant. The basic principle in theconstruction of genetically modified plants is to insert geneticinformation in the plant genome so as to obtain a stable maintenance ofthe inserted genetic material.

Several techniques exist for inserting the genetic information, the twomain principles being direct introduction of the genetic information andintroduction of the genetic information by use of a vector system. Areview of the general techniques may be found in articles by Potrykus(Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou(Agro-Food-Industry Hi-Tech March/April 1994 17-27).

Thus, in one aspect, the present invention relates to a vector systemwhich carries a nucleotide sequence or construct according to thepresent invention and which is capable of introducing the nucleotidesequence or construct into the genome of an organism, such as a plant.

The vector system may comprise one vector, but it can comprise twovectors. In the case of two vectors, the vector system is normallyreferred to as a binary vector system. Binary vector systems aredescribed in further detail in Gynheung An et al. (1980), BinaryVectors, Plant Molecular Biology Manual A3, 1-19.

One extensively employed system for transformation of plant cells with agiven nucleotide sequence is based on the use of a Ti plasmid fromAgrobacterium tumefaciens or a Ri plasmid from Agrobacterium rhizogenesAn et al. (1986), Plant Physiol. 81, 301-305 and Butcher D. N. et al.(1980), Tissue Culture Methods for Plant Pathologists, eds.: D. S.Ingrams and J. P. Helgeson, 203-208.

Several different Ti and Ri plasmids have been constructed which aresuitable for the construction of the plant or plant cell constructsdescribed above. A non-limiting example of such a Ti plasmid is pGV3850.

The nucleotide sequence or construct of the present invention shouldpreferably be inserted into the Ti-plasmid between the terminalsequences of the T-DNA or adjacent a T-DNA sequence so as to avoiddisruption of the sequences immediately surrounding the T-DNA borders,as at least one of these regions appear to be essential for insertion ofmodified T-DNA into the plant genome.

As will be understood from the above explanation, if the organism is aplant, then the vector system of the present invention is preferably onewhich contains the sequences necessary to infect the plant (e.g. the virregion) and at least one border part of a T-DNA sequence, the borderpart being located on the same vector as the genetic construct.Preferably, the vector system is an Agrobacterium tumefaciens Ti-plasmidor an Agrobacterium rhizogenes Ri-plasmid or a derivative thereof, asthese plasmids are well-known and widely employed in the construction oftransgenic plants, many vector systems exist which are based on theseplasmids or derivatives thereof.

In the construction of a transgenic plant the nucleotide sequence orconstruct of the present invention may be first constructed in amicroorganism in which the vector can replicate and which is easy tomanipulate before insertion into the plant. An example of a usefulmicroorganism is E. coli., but other microorganisms having the aboveproperties may be used. When a vector of a vector system as definedabove has been constructed in E. coli. it is transferred, if necessary,into a suitable Agrobacterium strain, e.g. Agrobacterium tumefaciens.The Ti-plasmid harbouring the nucleotide sequence or construct of thepresent invention is thus preferably transferred into a suitableAgrobacterium strain, e.g. A. tumefaciens, so as to obtain anAgrobacterium cell harbouring the nucleotide sequence or construct ofthe present invention, which DNA is subsequently transferred into theplant cell to be modified.

In this way, the nucleotide or construct of the present invention can beintroduced into a suitable restriction position in the vector. Thecontained plasmid is used for the transformation in E. coli. The E. colicells are cultivated in a suitable nutrient medium and then harvestedand lysed. The plasmid is then recovered. As a method of analysis thereis generally used sequence analysis, restriction analysis,electrophoresis and further biochemical-molecular biological methods.After each manipulation, the used DNA sequence can be restricted andconnected with the next DNA sequence. Each sequence can be cloned in thesame or different plasmid.

After each introduction method of the desired nucleotide sequenceaccording to the present invention in the plants the presence and/orinsertion of further DNA sequences may be necessary. If, for example,for the transformation the Ti- or Ri-plasmid of the plant cells is used,at least the right boundary and often however the right and the leftboundary of the Ti-and Ri-plasmid T-DNA, as flanking areas of theintroduced genes, can be connected. The use of T-DNA for thetransformation of plant cells has been intensively studied and isdescribed in EP-A-120516; Hoekema, in: The Binary Plant Vector SystemOffset-drukkerij Kanters B. B., Alblasserdam, 1985, Chapter V; Fraley,et al., Crit. Rev. Plant Sci., 4:1-46; and An et al., EMBO J. (1985)4:277-284.

Direct infection of plant tissues by Agrobacterium is a simple techniquewhich has been widely employed and which is described in Butcher D. N.et al. (1980), Tissue Culture Methods for Plant Pathologists, eds.: D.S. Ingrams and J. P. Helgeson, 203-208. For further teachings on thistopic see Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991]42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 199417-27). With this technique, infection of a plant may be done on acertain part or tissue of the plant, i.e. on a part of a leaf, a root, astem or another part of the plant.

Typically, with direct infection of plant tissues by Agrobacteriumcarrying the nucleotide sequence, a plant to be infected is wounded,e.g. by cutting the plant with a razor or puncturing the plant with aneedle or rubbing the plant with an abrasive. The wound is theninoculated with the Agrobacterium. The inoculated plant or plant part isthen grown on a suitable culture medium and allowed to develop intomature plants.

When plant cells are constructed, these cells may be grown andmaintained in accordance with well-known tissue culturing methods suchas by culturing the cells in a suitable culture medium supplied with thenecessary growth factors such as amino acids, plant hormones, vitamins,etc. Regeneration of the transformed cells into genetically modifiedplants may be accomplished using known methods for the regeneration ofplants from cell or tissue cultures, for example by selectingtransformed shoots using an antibiotic and by subculturing the shoots ona medium containing the appropriate nutrients, plant hormones, etc.

Further teachings on plant transformation may be found in EP-A-0449375.

Production of the Polypeptide

According to the present invention, the production of the polypeptide ofthe present invention can be effected by the culturing of, for example,microbial expression hosts, which have been transformed with one or morepolynucleotides of the present invention, in a conventional nutrientfermentation medium. The selection of the appropriate medium may bebased on the choice of expression hosts and/or based on the regulatoryrequirements of the expression construct. Such media are well-known tothose skilled in the art. The medium may, if desired, contain additionalcomponents favouring the transformed expression hosts over otherpotentially contaminating microorganisms.

Antibodies

The amino acid sequence of the present invention can also be used togenerate antibodies—such as by use of standard techniques—against theamino acid sequence. For the production of antibodies, various hostsincluding goats, rabbits, rats, mice, etc. may be immunized by injectionwith the inhibitor or any portion, variant, homologue, fragment orderivative thereof or oligopeptide which retains immunogenic properties.Depending on the host species, various adjuvants may be used to increaseimmunological response. Such adjuvants include, but are not limited to,Freund's, mineral gels such as aluminium hydroxide, and surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG(Bacilli Calmette-Guerin) and Corynebacterium parvum are potentiallyuseful human adjuvants which may be employed.

Monoclonal antibodies to the amino acid sequence may be even preparedusing any technique which provides for the production of antibodymolecules by continuous cell lines in culture. These include, but arenot limited to, the hybridoma technique originally described by Koehlerand Milstein (1975 Nature 256:495-497), the human B-cell hybridomatechnique (Kosbor et al (1983) Immunol Today 4:72; Cote et al (1983)Proc Natl Acad Sci 80:2026-2030) and the EBV-hybridoma technique (Coleet al (1985) Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc,pp 77-96). In addition, techniques developed for the production of“chimeric antibodies”, the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity can be used (Morrison et al (1984) Proc NatIAcad Sci 81:6851-6855; Neuberger et al (1984) Nature 312:604-608; Takedaet al (1985) Nature 314:452-454). Alternatively, techniques describedfor the production of single chain antibodies (U.S. Pat. No. 4,946,779)can be adapted to produce inhibitor specific single chain antibodies.

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inOrlandi et al (1989, Proc Natt Acad Scd 86. 3833-3837), and Winter G andMilstein C (1991; Nature 349:293-299).

PROTOCOLS Protocol 1 Xylanase Assay

(Endo-β-1,4-Xylanase Activity)

Xylanase samples are diluted in citric acid (0.1M)—di-sodium-hydrogenphosphate (0.2M) buffer, pH 5.0, to obtain approx. OD=0.7 in the finalassay. Three dilutions of the sample and an internal standard with adefined activity are thermostated for 5 minutes at 40° C. To time=5minutes, 1 Xylazyme tab (crosslinked, dyed xylan substrate) is added tothe enzyme solution. To time=15 minutes the reaction is terminated, byadding 10 ml of 2% TRIS. The reaction mixture is centrifuged and the ODof the supernatant is measured at 590 nm. Taking into account thedilutions and the amount of xylanase, the activity (TXU,Total-Xylanase-Units) of the sample can be calculated relatively to thestandard.

Protocol 2 Stickiness Protocol

(Stickiness Determination)

Dough stickiness is measured on a TA-XT2 system (Stable Micro Systems)using a SMS Dough Stickiness Cell. The protocol is a modified version ofthe method described by Chen and Hoseney (1995). A dough is made fromflour, 2% NaCl and water to 400 Brabender Units (BU) using a Farinograph(AACC method 54-21). The flour and NaCl is dry mixed for 1 minute. Wateris added and the dough is mixed for another 5 minutes. The obtaineddough could advantageously be rested for 10, 30 or 45 minutes in sealedcontainers at 30° C.

Approx. 4 gram dough is placed in the Dough Stickiness Cell. 4 mm doughis extruded to obtain an uniform extrusion. Hereafter 5 measurement aremade according to Stable Micro Systems protocol (TA-XT2 applicationstudy for measurement of dough stickiness). In brief, 1 mm dough isextruded. The probe (25 mm perspex cylinder probe), connected to theTA-XT2 system, is pressed into the extruded dough at a set force. Theprobe is raised and the adhesion between the dough and the probe isrecorded. The following TA-XT2 setting are used: Option: Adhesive testPre-test speed:  2.0 mm/s Test speed:  2.0 mm/s Post-test speed: 10.0mm/s Distance:   15 mm Force:   40 g Time:  0.1 s Trigger Type: Auto - 5g Data Acquisition rate:  400 pps

The results recorded from the test are peak force, meaning the forceneeded to raise the probe from the extruded dough. The distance, meaningthe distance the dough attach to the probe. Area, meaning area below theobtained curve.

Dough stickiness is depending on the quality of the flour used and therecipe. Therefore a non-sticky dough is a dough differing in stickinessfrom 100% to 200% (relative) compared to a reference dough, without thexylanase or having preferably less than 70% (relative) of the stickinessobtained with a commercial fungal xylanase (i.e. Pentopan mono BG, NovoNordisk) when dosed at a levels giving the same volume increase in abaking trial.

Protocol 3 Inhibitor Assay Protocol

(Inhibitor Assay)

To detect the inhibitor during isolation and characterisation thefollowing assay is used. 100 μl inhibitor fraction, 250 μl xylanasesolution (containing 12 TXU/ml) and 650 μl buffer (0.1 M citricacid—0.2M di-sodium hydrogen phosphate buffer, pH 5.0) is mixed. Themixture is thermostated for 5 minutes at 40.0° C. At time=5 minutes oneXylazyme tab is added. At time=15 minutes the reaction is terminated byadding 10 ml 2% TRIS. The reaction mixture is centrifuged (3500 g, 10minutes, room temperature) and the supernatant is measured at 590 nm.The inhibition is calculated as residual activity compared to the blank.The blank is prepared the same way, except that the 100 μl inhibitor issubstituted with 100 μl buffer (0.1 M citric acid—0.2 M di-sodiumhydrogen phosphate buffer, pH 5.0). By way of example, XM-1 may beconsidered to have a high degree of resistance to the inhibitor (seeFIG. 20). XM-2 and XM-3 may be considered to have a medium degree ofresistance to the inhibitor (see FIG. 20).

Protocol 4 Glucanase Protocol I

(Endo-β-1,4-Glucanase Activity)

Glucanase samples are diluted in 0.1M sodium-acetate—citric acid buffer,pH=5.0, to obtain approx. OD=0.7 in the final assay. Three dilutions ofthe sample and an internal standard with a defined activity arethermostated for 5 minutes at 40° C. To time=5 minutes, 1 Glucazyme tab(crosslinked, dyed glucan substrate) is added to the enzyme solution. Totime=15 minutes the reaction is terminated, by adding 10 ml of 2% TRIS.The reaction mixture is centrifuged and the OD of the supernatant ismeasured at 590 nm. Taking into account the dilutions and the amount ofglucanase, the activity (BGU, Beta-Glucanase-Units) of the sample can becalculated relatively to the standard.

Protocol 5 Inhibitor Assay Protocol II

(Inhibitor Kinetics Assay)

To study kinetics on the inhibitor a soluble substrate was used(Azo-xylan, Megazyme). A 2% (w/v) solution of the substrate wasprepared, according to manufacturers protocol, in 20 mM NaPi, pH 6.0.The assay was performed by pre-heating substrate, xylanase and inhibitorat 40° C. for 5 minutes.

For a preliminary inhibitor characterisation, the xylanase used isdiluted to 40 TXU/ml. For K_(i) determinations, the xylanases arediluted to approx. 40 TXU/ml.

0.5 ml of substrate, 0.1 ml of xylanase and 0.1 ml of inhibitor wasmixed at time=0 minutes, 40° C. At time=125 minutes, the reaction wasterminated by adding 2 ml of ethanol (95%), followed by vortexing for 10seconds. Precipitated unhydrolysed substrate was removed bycentrifugation (3500× g, 10 minutes, room temperature). OD in thesupernatant was measured against water at 590 nm.

A blank was prepared the same way. The only modification wassubstitution of the inhibitor with 20 mM NaPi, pH 6.0.

For kinetic experiments with decreased substrate concentration, thefollowing substrate concentrations were made by dilution in 20 mM NaPi,pH 6.0. 2%, 1%, 0.5% and 0.25% soluble azo-xylan (w/v).

For K_(i) determinations the above mentioned xylanases and substrateconcentrations were used. These were combined with the followingconcentrations of inhibitor extract in the assay: 0, 2, 5, 10, 25, 50and 100 μl in the assay. Using μl inhibitor and not a molarconcentration of the inhibitor, K_(i) is expressed as μl inhibitor.

SUMMARY

In summary the present invention provides inter alia:

-   -   a. The isolation of an endogenous endo-β-1,4-xylanase inhibitor        from wheat flour.    -   b. The characterisation of an endogenous endo-β-1,4-xylanase        inhibitor isolated from wheat flour.    -   c. The characterisation of the effect of endogenous        endo-β-1,4-xylanase inhibitor on different xylanases.    -   d. A means for selecting xylanases not detrimentally affected by        endogenous endo-β-1,4-xylanase inhibitor.    -   e. A means for selecting xylanases which are not detrimentally        affected by endo-β-1,4-xylanase inhibitors.    -   f. Xylanases that provide dough exhibiting favourable volume and        acceptable stickiness than when compared to doughs comprising        fungal xylanases.    -   g. A method for screening xylanases and/or mutating the same        using an endogenous endo-β-1,4-xylanase inhibitor, and the use        of those xylanases or mutants thereof in the manufacture of        doughs.    -   h. A foodstuff prepared with the xylanases of the present        invention.

DEPOSITS

The following samples were deposited in accordance with the BudapestTreaty at the recognised depositary The National Collections ofIndustrial and Marine Bacteria Limited (NCIMB) at 23 St. Machar Drive,Aberdeen, Scotland, United Kingdom, AB2 1RY on 22 December 1998:DH5α::pCR2.1_BS xylanase NCIMB number NCIMB 40999 BL21(DE3)::pET24A_XM1NCIMB number NCIMB 41000 BL21(DE3)::pET24A_XM3 NCIMB number NCIMB 41001DH5α::pCR2.1_BS xylanase comprises wild type xylanase.BL21(DE3)::pET24A_XM1 comprises XM1 xylanase.BL21(DE3)::pET24A_XM3 comprises XM3 xylanase.

The present invention also encompasses sequences derivable and/orexpressable from those deposits and embodiments comprising the same.

INTRODUCTION TO THE EXAMPLES SECTION AND THE FIGURES

The present invention will now be described, by way of example only,with reference to the accompanying drawings in which:

FIG. 1 shows a graph;

FIG. 2 shows a graph;

FIG. 3 shows a graph;

FIG. 4 shows a graph;

FIG. 5 shows a graph;

FIG. 6 shows a graph;

FIG. 7 shows a graph;

FIG. 8 shows a graph;

FIG. 9 shows a graph;

FIG. 10 shows a graph;

FIG. 11 shows an image result of an SDS PAGE experiment;

FIG. 12 shows a graph;

FIG. 13 shows a graph;

FIG. 14 shows a graph;

FIG. 15 shows a graph;

FIG. 16 shows a graph;

FIG. 17 shows an image result of an IEF experiment;

FIG. 18 shows a graph;

FIG. 19 shows a graph;

FIG. 20 shows a graph;

FIG. 21 shows a graph;

FIG. 22 shows a graph;

FIG. 23 shows a graph;

FIG. 24 shows a graph;

FIG. 25 shows a graph;

FIG. 26 shows a graph;

FIG. 27 shows a graph;

FIG. 28 shows a graph;

FIG. 29 shows a graph;

FIG. 30 shows a graph; and

FIG. 31 shows a graph.

In slightly more detail:

FIG. 1—Stickiness as a function of xylanases, dose and resting time.

FIG. 2—Stickiness as a function of xylanases, dose and resting time.

FIG. 3—Gel filtration chromatography of a 75 ml inhibitor extractsample. Column: 500 ml Superdex G-25 F, Flow: 10 ml/min, Fraction size:30 ml.

FIG. 4—Cation exchange chromatography of a 240 ml gel filtratedinhibitor extract sample. Column: 50 ml Sepharose SP, Flow: 5.0 ml/min,Fraction size: 10 ml.

FIG. 5—HIC chromatography of a 147 ml ion exchanged inhibitor extractsample added (NH₄)₂SO₄ to 1.0M. Column: 10 ml Phenyl HIC, Flow: 2.0ml/min, Fraction size: 2.5 ml.

FIG. 6—Preparative gel filtration chromatography of 2 ml concentratedinhibitor sample. Inhibitor eluted at 176 ml. Column: 330 ml Superdex 75PG (Pharmacia). Eluent: 50 mM NaOAc, 200 mM NaCl, pH 5.0. Flow: 1ml/minute. Fraction size: 5.5 ml.

FIG. 7—Cation exchange chromatogram of pure xylanase+boiled inhibitorextract. Sample: 1 ml desalted 980601+boiled inhibitor extract. Column:1 ml Source S 15. Buffer system: A: 50 mM NaOAc, pH 4.5, B: A+1 M NaCl.Flow: 2 ml/minute.

FIG. 8—Cation exchange chromatogram of pure xylanase after three hoursincubation with inhibitor extract. Sample: 1 ml desalted980601+inhibitor. Column: 1 ml Source S 15. Buffer system: A: 50 mMNaOAc, pH 4.5, B: A+1 M NaCl. Flow: 2 ml/minute.

FIG. 9—Analytical gel filtration chromatography of 100 μl concentratedinhibitor sample. Inhibitor eluted at 10.81 ml. Column: 24 ml Superdex75 10/30 (Pharmacia, Sweden). Eluent: 50 mM NaOAc, 100 mM NaCl, pH 5.0,Flow: 0.5 ml/minute. Fraction size: 2.0 ml.

FIG. 10—Log(MW) as function of Kav for standard proteins run on aSuperdex 75 10/30.

FIG. 11—SDS PAGE of fraction 31, 32 and fraction 33 from Preparative gelfiltration. Lane 1 and 3 are MW markers (Pharmacia's LMW markers,Sweden). Lane 2 and 4 are frac. 32, loaded with 10 and 25 μlrespectively. Lane 6 and 8 are frac. 31, loaded with 10 and 25. Lane 7and 9 are frac. 33, loaded with 10 and 25.

FIG. 12—Reverse Phase Chromatogram of fraction 33 from Gel FiltrationChromatography. Chromatogram reveals four destinct peaks. Peak 3 is thexylanase inhibitor. Peak 4, 5 and 6 are sequenced and show very highhomology to the Wheat protein, Serpin.

FIG. 13—MS of fraction 3 from RP—chromatography. Spectra shows onemolecule having a molecular weight of 39503 Da.

FIG. 14—Reverse Phase Chromatography of carboxy methylated fraction 3from Reverse Phase Chromatogram of fraction 33 (see FIG. 12). Thechromatogram revealed two destinct peaks (fraction 2 and 3), indicatinga di-peptide.

FIG. 15—MS of fraction 2 from carboxy methylated Reverse PhaseChromatography (see FIG. 14). Spectra indicate a peptide having amolecular weight of 12104 Da.

FIG. 16—MS of fraction 3 from carboxy methylated Reverse PhaseChromatography (see FIG. 14). Spectra indicate a peptide having amolecular weight of 28222 Da.

FIG. 17—IEF of fraction 33 and 34 from Preperative Gel FiltrationChromatography. Lane 2 is pl 3-10 standards, lane 3 is p l 2.5-6.5standards, lane 4 and 5 are fraction 33 and 34—respectively, lane 6 isTrysin Inhibitor (pl 4.55), lane 7 is β-lactoglobulin (pl 5.20) and lane8 and 9 are are fraction 33 and 34—respectively. Arrows indicatedestinct bands in fraction 33.

FIG. 18—pH and relative OD (from inhibitor assay) as function offractions from Chromatofocusing Chromatography of xylanase inhibitor. Ascan be seen from the figure, the relative OD decreases in fraction 7,indicating inhibitor activity. This correspond to pH 9.4.

FIG. 19—Residual activity, % of four xylanases as a function ofinhibitor concentration. The four xylanases used are -♦-X1, -▪-X3,-x-BX, -▴-Novo.

FIG. 20—Residual activity of 980601 (coli-1)_(,) 980603 (Belase) andthree mutants of 980601 (XM1, XM2 and XM3) after incubation with a flourextract.

FIG. 21—Line-weaver—Burk plot of xylanase (980601) +/− inhibitor.Substrate concentration is % azo-xylan. V is relative OD 590 from assay(where 100 is S=2%).

FIG. 22—K_(i) for different xylanases expressed as microliter inhibitor.

FIG. 23—Inhibition of three xylanases (980601=Bac. sub. wt, 980801=X1and 980901=Thermomyces) as a function of pH. The data are obtained bysubstrating relevant blanks.

FIG. 24—pH optimum for three xylanases (980601=BX, 980801=X1 and980901=Novo).

FIG. 25—Spec. vol=f(xylanase x dose)

FIG. 26—Spec. vol. increase=f(xylanase x dose)

FIG. 27—Stickiness=f(xylanase x dose)

FIG. 28—Stickiness as function of different xylanase preparations andcontrol, measured after 10 (_(—)10) and 45 (_(—)45) minutes resting.980603 is purified Röhm xylanase, XM1 is xylanase mutant 1 and #2199 isRöhm's Veron Special product.

FIG. 29—Stickiness increase as function of three xylanase preparations,after 10 (_(—)10) and 45 (_(—)45) minutes resting. 980603 is purifiedRöhm xylanase, XM1 is xylanase mutant 1 and #2199 is Röhm's VeronSpecial product.

FIG. 30—Stickiness increase as function of two xylanase preparations,after 10 (_(—)10) and 45 (_(—)45) minutes resting. XM1 is xylanasemutant 1 and #2199 is Röhm's Veron Special product.

FIG. 31—Stickiness increase as function of added Endo-β-1,4-Glucanase.1: Control dough without xylanase, 2:7500 TXU pure Röhm xylanase/kgflour, 3:7500 TXU pure Röhm xylanase/kg flour+158 BGU/kg Flour, 4:15000TXU pure Röhm xylanase/kg flour, 5:15000 TXU pure Röhm xylanase/kgflour+316 BGU/kg Flour. Dough were measured after 10 (Stik_(—)10) and 45(Stik_(—)45) minutes.

EXAMPLES Example 1

Dough Stickiness as a Function of Different Xylanases, Doses and RestingTime

The following xylanases ability to give dough stickiness were tested.

(See also Chen, W. Z. and Hoseney, R. C. (1995). Development of anobjective method for dough stickiness. Lebensmittel Wiss u.- Technol.,28, 467-473.)

Enzymes

“X1” corresponds to a purified sample of endo-β-1,4-xylanase fromAspergillus niger. This xylanase has an activity of 8400 TXU (15000TXU/mg).

“Novo” corresponds to Novo Nordisk's Pentopan Mono BG from Thermomyces.This xylanase has an activity of 350.000 TXU (56000 TXU/mg).

“BX” corresponds to a purified sample of the new bacterial xylanase.This sample has an activity of 2000 TXU (25000 TXU/mg).

“Röhm” corresponds to Röhm GmbH's bacterial xylanase, Veron Speciel.This sample has an activity of 10500 TXU (25000 TXU/mg).

Xylanase Assay

Xylanase assays were performed according to Protocol 1

Flour

Two kinds of flour have been used in this trial: Danish flour, batch no98022 and German flour, batch no. 98048. The water absorbtions, at 400BU, of the two kinds of flour are 58 and 60% respectively.

Dough Preparation

Dough were prepared as described in Protocol 2. After mixing the doughrested for 10 and 45 minutes respectively at 30° C. in sealedcontainers.

Stickiness Measurement

Stickiness measurements were performed according to Protocol 2

Results and Discussion

Fungal Xylanases Versus New Bacterial Xylanase

The following dough were made and tested for dough stickiness after 10and 45 minutes in flour 98048. TABLE 1 Dough made with different dosesof two fungal xylanases and one bacterial xylanase. (Dose is calculatedper kg of flour.) Enzyme TXU./kg Blank 0 X1 (980801) 1500 10000 Novo(#2165) 5000 60000 BX (980802) 1500 15000

The dough in Table 1 gave the dough stickiness results presented inTable 2 and FIG. 1. TABLE 2 Dough made with different doses of differentxylanases vs.blank. The dough was rested for 10 and 45 minutes,respectively. Stickiness is given as g × s, the stickiness figure is anaverage of 5 determinations. Stickiness, Dough g × s Std.Dev std.dev., %Control, 10 min 5.533 0.16 2.89 Control, 45 min 8.103 0.277 3.42  1500X1, 10 min 7.275 0.204 2.80  1500 X1, 45 min 8.675 0.134 1.54 10000 X1,10 min 9.295 0.802 8.63 10000 X1, 45 min 13.339 1.264 9.48  5000 Novo,10 min 6.757 0.218 3.23  5000 Novo, 45 min 7.23 0.337 4.66 60000 Novo,10 min 10.972 0.519 4.73 60000 Novo, 45 min 16.559 1.626 9.82  1500 BX,45 min 4.372 0.358 8.19 15000 BX, 10 min 6.567 0.639 9.73 15000 BX, 45min 5.545 0.518 9.34

The data from Table 2 are illustrated in FIG. 1.

As can be seen from Table 2 and FIG. 1 the fungal xylanase X1 and thexylanase in the Novo product give rise to dough stickiness. The newbacterial xylanase does not give rise to the same stickiness. Inaddition, the stickiness seems to decrease compared with control.

New Bacterial Xylanase vs Röhm's Bacterial Xylanase

To test the functionality of the novel bacterial xylanase compared tothe bacterial xylanase in the Röhm product: Veron Special, the followingdough was made (see Table 3) using flour 98022. TABLE 3 Dough made withdifferent doses of two bacterial xylanases. (Dose is calculated per kgof flour.) Enzyme TXU/kg Blank 0 BX 5000 15000 Röhm 5000 15000

The dough in Table 3 gave the dough stickiness results presented inTable 4 and FIG. 2. TABLE 4 Dough made with different doses of differentxylanases vs. blank. Stickiness is given as g × s, the stickiness figureis an average of 5 determinations. Stickiness, Dough g × mm Std.Devstd.dev,. % Control 10 min 5.269 0.16 3.04 Control 45 min 5.484 0.2775.05  5000 BX, 10 min 4.443 0.204 4.59  5000 BX, 45 min 4.474 0.134 3.0015000 BX, 10 min 4.791 0.352 7.35 15000 BX, 45 min 6.288 0.599 9.53 5000 Röhm, 10 min 5.077 0.218 4.29  5000 Röhm, 45 min 6.757 0.337 4.9915000 Röhm, 10 min 7.749 0.519 6.70 15000 Röhm, 45 min 10.98 0.907 8.26

The data from Table 4 are illustrated in FIG. 2.

The results show that BX (the new bacterial xylanase) gives rise to muchless stickiness than the fungal xylanase tested. Moreover, it is foundthat the new xylanase gives rise to much less dough stickiness than theRöhm bacterial xylanase.

Example 2

Inhibitor Purification, Characterisation and Effect on Xylanases

Flour

Three different kinds of flour was used in these experiments (batch98002, 98026 and 98058). Flour batch 98002 and 98058 is Danish flour.Flour batch 98026 is German flour.

Inhibitor Extraction

The inhibitor was extracted from the flour using ice cold distilledwater and stirring. One equivalent of flour was added two equivalents ofice cold distilled water. The mix was added a magnetic bar, placed in anice bath and stirred for 20 minutes. After stirring the flour slurry waspoured into centrifuge vials and centrifuged (10000 g, 4° C. and 10minutes). The supernatant contained the xylanase inhibitor.

Inhibitor Assay

Inhibitor assays were performed according to Protocol 3

Inhibitor Isolation

After extraction of a 100 g flour sample (98026) the xylanase inhibitorwas purified by the following chromatographic techniques:

Gel Filtration Chromatography (This Procedure was Run Twice)

75 ml extract was applied to a 500 ml Superdex G-25 F (Pharmacia,Sweden) column at 10 ml/minute, calibrated with 20 mM NaOAc, pH 4.25.Eluent was collected in 30 ml fractions at the same flow. All fractionswere spotted for inhibitor.

Cation Exchange Chromatography (This Procedure was Run Twice)

The inhibitor peak collected from the gel filtration run (240 ml) wasapplied to a 50 ml SP Sepharose (Pharmacia, Sweden) column at 5ml/minute. After loading, the column was washed to baseline with Abuffer (20 mM NaOAc, pH 4.25). The inhibitor was eluted by a lineargradient from A to B buffer (B: A+350 mM NaCl) over 10 column volumes atthe same flow. The eluate was collected in fractions of 10 ml. Everysecond fraction was spotted for xylanase inhibitor.

Hydrophobic Interaction Chromatography (This Procedure was Run Twice)

The inhibitor peak from the cation exchange chromatography (110 ml) wasadded (NH₄)₂SO₄ to 1.0 M and applied to a 10 ml Phenyl Sepharose HIC(Pharmacia, Sweden) column at 2 ml/minute. The inhibitor was eluted fromthe column by a 12 column volume linear gradient from A (20 mM NaPi, 1 M(NH₄)₂SO₄, pH 6.0) to B (20 mM NaPi, pH 6.0). The eluate was collectedin fractions of 2.5 ml. Every second fraction was spotted for xylanaseinhibitor.

Preparative Gel Filtration Chromatography

5 ml inhibitor peak from HIC run was up-concentrated to 2 ml using arotatory evaporator. This sample was loaded to a 330 ml Superdex 75 PG(Pharmacia, Sweden) column at 1 ml/minute. The buffer system used was 50mM NaOAc, 0.2 M NaCl, pH 5.0. The eluate was collected in 5.5 mlfractions. Every second fraction was spotted for xylanase inhibitor.

Analysis of Protease Activity

To be able to determine whether the found inhibitor effect was due to aninhibitor or a protease hydrolysing the xylanase, the followingexperiments were carried out.

Incubation Trials

2 ml of pure xylanase, 980601 (see Endo-β-1,4-xylanases) was incubatedwith 0.25 ml of inhibitor extract for three hours at 40 degree C. As acontrol the same incubation was made with boiled (5 minutes) inhibitorextract. After incubation the samples were added 50 mM NaOAc, pH 4.5 to2.5 ml and desalted by gel filtration on a PD-10 column (Pharmacia,Sweden), obtaining 3.5 ml sample in 50 mM NaOAc, pH 4.5.

Analysis for Hydrolysis

The two samples of pure xylanase from the incubation trials wereanalysed on a SOURCE 15 S column. 1 ml of the gel filtered sample wasapplied to the column (calibrated with A buffer: 50 mM NaOAc, pH 4.5) at2 ml/minute. The sample was eluted with a linear gradient from A to B(B:A+1 M NaCl) over 20 column volumes and collected in 2 ml fractions.The xylanase was detected using OD 280 nm and spotted for xylanaseactivity in the fractions (100 μl fraction +900 μl buffer (0.1 M citricacid −0.2 M di-sodium hydrogene phosphate buffer, pH 5.0) +1 Xylazymetab, 10 minutes, 40 degree C. Reaction terminated with 10 ml 2% TRIS,blue colour=xylanase activity).

Inhibitor Characterisation

Analytical Gel Filtration Chromatography

100 μl (concentrated two times on rotatory evaporator) of the inhibitorpeak from the HIC run was applied to a 24 ml Superdex 75 10/30(Pharmacia, Sweden) at 0.5 ml/minute. Running buffer used was 50 mMNaOAc, 0.1 M NaCl, pH 5.0. Eluate was collected in fractions of 2 ml.All fractions were spotted for inhibitor.

To be able to determine the size of the inhibitor a series of knownproteins were applied to the 24 ml Superdex 75 10/30 column. Theconditions for this run were as described above. The standard proteinsused were: Protein Size, KDa. BSA 67 Ovalbumine 43 Chymotrypsine 25Ribonuclease A 13.7

The proteins were detected at 280 nm.

SDS PAGE

Fractions from Preparative gel filtration chromatography were added SDSsample buffer (prepared according to NOVEX protocol), boiled for threeminutes and loaded on a 8-16% PAGE gel (NOVEX). The gel was stainedaccording to NOVEX's protocol for silver staining. As molecular weightmarkers, Pharmacia's LMW markers were used.

Iso Electric Focusing (IEF)

To determine the pl of the native inhibitor, a sample of purifiedinhibitor (fraction 33 from 330 ml Superdex 75 PG) was loaded on a pH3-10 IEF GEL (NOVEX). The gel was run according to manufactors protocol.Using Pharmacia's (Sweden) Broad pl kit, 3.5-9.3 as standards. The gelwas stained with coomassie brilliant blue, according to producersprotocol.

Chromatofocusing Chromatography

A sample of fraction 33 from Preperative gelfiltration chromatography,was gelfiltrated to water. 100 μl desaltet sample was loaded on a Mono PHR 5/5 (Pharmacia, Sweden). Starting conditions was obtained with 25 mMethanolamin-HCl, pH 9.4. The column was eluted with Poly buffer 96:Waterina 1:10 ratio. pH adjusted to 6.0 (flow:0.5 ml/min; fraction size:0.5ml). After elution with Poly buffer 96, the column was further elutedwith Poly buffer 74:water in a 1:10 ratio. pH adjusted to 3.80 (flow:0.5ml/min; fraction size:0.5 ml).

All fractions was pH measured and spotted for xylanase inhibitor, usingProtocol 3.

Amino Acid Sequence

A sample (obtained from fraction 33 from 330 ml Superdex 75 PG) of pureinhibitor from preperative purification was used. 200 μl if was loadedon a C4 Reverse Phase column (Applied Biosystems). The buffersystem usedwas A:0.1% TFA in water and B:0.1% TFA in 100% Acetonitrile. Inhibitorpeak from this run was carboxymethylated and rerun on C4 column again.In this way two inhibitor peptides, of interest, were obtained. Thesewere N-terminal sequenced. Furthermore, the peptides were digested withLys-C. The obtained peptides were recovered using reverse phasechromatography and amino acid sequenced.

To verify sequnces obtained by amino acid sequencing, a small fractionof the sample of interest, was analysed using MS (Voyager).

Inhibitor Kinetics

Inhibitor assays were performed according to Protocol 5. In thisrespect, for the preliminary inhibitor characterisation studies, thexylanase used was 980601, diluted to 40 TXU/ml and the inhibitor wasextracted from flour 98002. For K_(i) determinations, the followingxylanases were used:980601, 980603, 980801, 980901, 980903, 980906 and980907, diluted to approx. 40 TXU/ml. The inhibitor used for K_(i)determinations was extracted from flour 98058.

Determination of Inhibition as a Function of pH

These experiments were carried out as described in Protocol 3 with thefollowing modifications. Besides using 650 μl buffer (0.1 M citric acid−0.2 M di-sodium hydrogene phosphate) pH 5.0 in the assay, the assay wasalso carried out using the same buffer system at pH:4, 6 and 7.

Endo-β-1,4-xylanases

The following xylanase preparations were used:

980601 (BX):Purified preparation of Danisco's new bacterial xylanaseexpressed in E. coli. (1225 TXU/ml)

980603 (Röhm):Purified preparation of Frimond's Belase xylanase(identical to Röhm's) (1050 TXU/ml)

980801 (X1):Purified X1 from Aspergillus niger (8400 TXU/g)

980802 (Röhm):Purified preparation of Frimond's Belase xylanase(identical to Röhm's) (265 TXU/ml)

980901 (Novo):Purified preparation of Thermomyces xylanase from Novo'sPentopan mono BG (2900 TXU/ml)

980903 (XM1):Purified mutant of Bacillus sub. wild type xylanaseexpressed in E. coli. (1375 TXU/ml)

980906 (XM3):Purified mutant of Bacillus sub. wild type xylanaseexpressed in E. coli. (1775 TXU/ml)

980907 (XM2):Purified mutant of Bacillus sub. wild type xylanaseexpressed in E coli. (100 TXU/ml)

9535 (X3):Purified xylanase, X3 from Aspergillus niger (6490 TXU/ml)

Results and Discussion

Inhibitor Extraction for Isolation and Characterisation

100 g flour (98026) was extracted. After centrifugation a supernatant of150 ml was obtained. The presence of inhibitor was checked in thisextract (Table 5) and found positive. TABLE 5 Residual activity as afunction of +/− addition of inhibitor extract from wheat flour (98026).The xylanase used is 980601. −inhibitor +inhibitor Residual activity, %OD 590 0.675 0.165 24.44Inhibitor Isolation

75 ml of the inhibitor extract was loaded on a 500 ml gel filtrationcolumn (FIG. 3). After spotting for the inhibitor, it could be locatedin fractions [4-11] (Table 6). TABLE 6 Fractions from gel filtrationchromatography of 75 ml inhibitor extract assayed for xylanaseinhibitor. OD run 1 respectively 2 correspond to the two runs that wereperformed on the column. Inhibitor was found present in fractions[4-11]. These fractions were pooled for each run, giving two times 240ml. Fraction no. OD run 1 OD run 2 1 0.674 2 0.665 3 0.652 4 0.618 0.4765 0.388 0.166 6 0.186 0.126 7 0.188 0.18 8 0.277 0.217 9 0.381 0.231 100.406 0.246 11 0.395 0.435 12 0.725 13 0.683 14 0.762 15 0.737

The pool of the inhibitor peak in both runs on the gel filtrationcolumn, was approx. 240 ml.

Two times, a 240 ml pool from gelfiltration was applied to the cationexchanger. The flow through was found negative for inhibitor. As can beseen from FIG. 4 and Table 7 the inhibitor bound to the column andeluted at approx. 750 mM NaCl. TABLE 7 Fractions from cation exchangechromatography of 240 ml gel filtered inhibitor extract assayed for thepresence of xylanase inhibitor. OD run 1 respectively 2 correspond tothe two runs that were performed on the column. Inhibitor was foundpresent in fractions [44-54]. Fraction no. OD run 1 OD run 2 40 0.4760.624 42 0.407 0.58 44 0.404 0.398 46 0.22 0.137 48 0.144 0.107 50 0.1980.126 52 0.302 0.208 54 0.395 0.435 56 0.457 0.495 58 0.463 0.606

The pool of inhibitor from the ion exchange runs was 110 ml from eachrun. These two pooled fractions were added (NH₄)₂SO₄ to 1.0 M andapplied to the HIC column in two runs. The flow through was spotted forinhibitor and found negative. As can be seen from FIG. 5 and Table 8 allinhibitor bound to the column and a good separation was obtained.

The analysis of the fractions from the HIC chromatography is shown inTable 8. TABLE 8 Fractions from HIC chromatography of 147 ml inhibitorextract assayed for xylanase inhibitor. OD run 1 respectively 2corresponds to the two runs that were performed on the column. Inhibitorwas found present in fractions [15-23]. Fraction no. OD run 1 OD run 2Blank 0.469 0.659 12 0.462 0.622 14 0.486 0.555 16 0.202 0.188 18 0.10.118 20 0.102 0.146 22 0.242 0.193 24 0.392 0.502 26 0.485 0.6

Fractions 17 and 18 from the HIC chromatography were concentratedapprox. two times and applied to a preparative gel filtration column(FIG. 6).

The analysis of the fractions from the preparative gel filtration isshown in Table 9. TABLE 9 Fractions from Preparative gel filtrationchromatography of 2 ml concentrated inhibitor sample assayed for thepresence of xylanase inhibitor. Inhibitor was found present in fractions[31-33]. Fraction no. OD 590 nm 26 0.738 28 0.774 30 0.645 32 0.117 340.705 36 0.749 38 0.754 40 0.761 42 0.769Analysis of Protease Activity

Based on the above assay of the xylanase inhibitor, it can not be ruledout that the decrease in xylanase activity, when mixed with the flourextract, is not due to a proteolytic hydrolysis of the xylanase.Therefore, a purified xylanase was incubated with an “inhibitor”extract. As can be seen from FIG. 7 and 8, no hydrolysis seems to occur.There is little more back-ground in the chromatogram with activeinhibitor (FIG. 8). However, this back-ground corresponds to thechromatogram of the inhibitor alone (chromatogram of inhibitor notshown). The difference in background must be due to precipitation in theboiled inhibitor sample.

Inhibitor Characterization

Analytical Gel Filtration Chromatography

100 μl two times concentrated inhibitor sample from fraction 18 in thesecond HIC run was applied to a 24 ml analytical Superdex 75 10/30(Pharmacia, Sweden) (FIG. 9). The eluate was collected in fractions of 2ml. These fractions were assayed for the xylanase inhibitor (Table 10).TABLE 10 Fractions from analytical gel filtration chromatography of 100μl concentrated inhibitor sample assayed for the presence of xylanaseinhibitor. Inhibitor was found present in fractions [6-7]. Fraction no.OD 590 nm Blank 0.613 6 0.233 7 0.304 8 0.51 9 0.569 10 0.565 11 0.652

After the gel filtration of the up-concentrated inhibitor sample a mixof four standard molecular weight proteins was applied to the column,using the exactly same procedure (chromatogram not shown). In table 11the molecular weights and the elution times for the proteins aresummarised. TABLE 11 Standard proteins used for determination of the MWof the inhibitor. Abbreviations and equations used are explained belowthe table. Std. protein Ve, ml Kav* MW, kDa log (MW) BSA 9.46 0.05950867 1.826075 Ovalbumin 10.38 0.119017 43 1.633468 Chymotrypsin 12.490.255498 25 1.39794 Ribonuklease A 13.49 0.320181 13.7 1.136721*Kav = (Ve − Vo)/Vt − Vo)Where:Ve = ret. Time, ml =Vo = void vol., ml = 8.54Vt = 24 ml = 24

Plotting the log (MW) as a function of Kav. It is possible to obtain anequation and estimating the molecular size of an unknown molecule (FIG.10).

Using the equation obtained in FIG. 10 and the retention time for theinhibitor, it is possible to calculate the molecular size of theinhibitor $\begin{matrix}{{MW},{{kDa} = 10^{({{- 2},{{4485 \times {kAV}} + 1.9602}})}}} \\{= 10^{({{- 2},{{4485 \times 0.173559} + 1.9602}})}} \\{= 10^{1.5352}} \\{= 34.29}\end{matrix}$

The molecular weight found for the inhibitor was higher than we expectedaccording to Rouau and Surget (1998, Evidence for the presence of aPentosanase Inhibitor in Wheat Flour. Journal of Cereal Science. 28:63-70)), the MW of the molecule is approx. 8 KDa. The MW obtained by gelfiltration could be explained by aggregation of several inhibitormolecules. To study this further an SDS PAGE gel was run of fractions31, 32 and 33 from the preparative gel filtration chromatography (FIG.11). As can be seen from this gel, three bands appears in the lanasloaded with purified inhibitor sample. These band correspond to proteinswith MW's of approx. 40, 30 and 10 kDa.

MW Determination using MS

A sample of fraction 33 from preparative gel filtration of the inhibitorwas desalted using the Presorb system and 5 volumes of 20 mM Aceticacid. 200 μl was loaded on a C4 Reverse Phase column (AppliedBiosystems). From this run, three peaks was obtained. One of these peaks(peak 3) was clearly dominating, and thought to be the inhibitor (FIG.12). The other peaks from the run have also been sequenced. From thesequence obtained it can be concluded that they are all originating fromthe same wheat protein, Serpin, and are not identical to the inhibitor(peak 3). Therefore peak 3 is concluded to be the xylanase inhibitor ofinterest. This peak was further characterised using MS (Voyager).

MS spectra analysis revealed a signal corresponding to a protein of39503 Da, using sinapic acid as matrix (FIG. 13).

As mentioned above, the SDS PAGE gel indicated three bands. One band atapprox. 10 kDa, one at approx. 30 kDa and a band at approx. 40 kDa. Toexplain the results seen from SDS-PAGE, the pure dominant fraction wascollected, lyophilised and carboxymethylated and then rerun on the C4column, using same conditions as mentioned above.

The fraction obtained by this rerun (FIG. 14) was analysed using MS. Ascan be seen from FIG. 15 and 16, the MW of these poly-peptides are 12104and 28222 Da.

Without wishing to be bound by theory we believe that the xylanaseinhibitor is either a native di-peptide (MW 39503 Da) or it isdenaturated and reduced (two peptides with MW 12104 and 28222Da—respectively) during the analytical process.

Determination of pl for the Xylanase Inhibitor using IEF andChromatofocusing Chromatography

The IEF gel showed three bands in the alkaline area (approx. 9.3, 8.6and 8.2—respectively) and three bands in the acidic area (approx. 5.1,5.3 and 5.5—respectively) (FIG. 17). Based on these results alone it maynot be feasible to determine the pl of the native xylanase inhibitor. Inthis regard, we knew from the sequencing results, that the sample onlycontained the xylanase inhibitor and three fracments of Serpin, ofapprox. 4500 Da. A theoritical calculation of the pi for Serpin is 5.58and the pl calculated on the fracment we obtained by sequencing gives pi5.46 (using Swiss Prot programmes). This could indicate that the threeacidic bands seen on the gel, are the three peaks of Serpin seen withReverse Phase Chromatography (FIG. 12), and the three alkaline bands arethe three different forms of the xylanase inhibitor, i.e. the nativedi-peptide form and the two peptides (as indicated by sequencing).

As can be seen from the Chromatofocusing Chromatography resultspresented in FIG. 18, the xylanase inhibitor does not bind to the columnunder the given conditions. This could mean that the native xylanaseinhibitor has a pi of 8.5 or even higher. Hence, it would seem that theasumptions presented above, namely that there are three alkaline bandson the IEF gel and so there could be three possible forms of thexylanase inhibitor, may be correct.

In conclusion, we believe that the native xylanase inhibitor has pl inthe interval 8.0-9.5. Within this interval, there are three bands. Thesethree bands probably correspond to the xylanase inhibitor possiblyexisting in three forms (see the results determined using IEF). In thisrespect, in using IEF, the protein runs as a native protein but thatsome di-peptide proteins may be partly damaged by this technique,thereby giving rise to more than one band.

Sequence Data

The two peptides forming the inhibitor, were sequenced giving N-terminaland internal sequences. The results are presented in the attachedsequence listings as SEQ ID No.s 13-19.

The sequences making up the first chain (chain A) are shown as SEQ IDNo.s 13 and 14. The sequences making up the second chain (chain B) areshown as SEQ ID No.s 15 to 19.

A data base search for homology to the sequenced poly-peptides came outnegative. Neither of the poly-peptides have been sequenced or describedbefore.

Effect of Inhibitor on Different Xylanases

Several trials have been carried out to study the inhibition ofdifferent xylanases. First we believed that the decrease in xylanaseactivity was due to a proteolytic activity in the extract. Therefore,different xylanases were incubated with different volumes of “inhibitor”extract (FIG. 19). The xylanases were found to be inhibited to differentextends. What we also found was that there seemed to be an increase ininhibition as a function of “inhibitor” concentration.

The results illustrated in FIG. 19 could indicate that the decrease wasdue to proteolysis or inhibitor. However, time course experiments withconstant xylanase and inhibitor concentrations and the above mentionedresults under “Analysis for protease activity”, did not show decreasedactivity as a function of time. To be able to distinguish betweenprotease and inhibitor, real kinetics has to be made (see “inhibitorkinetics”).

Two Bacillus subtilis xylanases have been studied very closely regardingtheir baking performance. These xylanases differed a little in theirfunctionality, meaning that one gave a slightly higher specific volumewhen baked in identical doses. One explanation could be differentinhibition of their activity in the flour. An experiment was thereforeperformed to examine this. The experiment has been repeated twice, usingtwo different kinds of flour as source for the inhibitor (Table 14).TABLE 14 inhibition of two xylanases (980601 and 980603) by inhibitorextracted from two kinds of flour (98002 and 98026). Inhibition iscalculated as % inhibition and as % residual activity, compared toblank. Folur 98002 98026 Inhibation, % 980601 67.03 75.04 980603 60.7661.33 98002 98026 Avg Rest act., % 980601 32.97 24.96 28.96 980603 39.2438.67 38.96 Difference, % 25.65

The trial shows that the two xylanases are inhibited to differentextents by the inhibitor. The xylanases differ in only six amino acids.

Based on 980601, three xylanase mutants have been made (XM1, XM2 andXM3). These mutants have been analysed for inhibition (FIG. 20).

As can be seen from FIG. 20 the three mutants differ in residualactivity, meaning that they are inhibited to different degrees by thexylanase inhibitor. Four (BX, Röhm, XM1 and XM3) of the five xylanaseshave the same specific activity (approx. 25000 TXU/mg protein). XM2 isexpected to have the same specific activity.

The difference in inhibition between XM1 and XM2 is approx. 250% (theresidual activity of XM1 is 2.5 times higher than the rest activity ofXM2). This difference is due to one amino acid. Amino acid 122 in XM2 ischanged from arginine to asparagine, introducing less positive chargnear the active site.

Inhibitor Kinetics

Simple preliminary kinetics were performed. Just to be able to determinewhether the inhibitor is competitive or non-competitive.

Different amounts of substrate were incubated with a constant xylanase-and inhibitor concentration (FIG. 21).

As can be seen from FIG. 21, V_(max) for both xylanase with and withoutinhibitor is approx. 1.19. This indicates that the inhibition iscompetitive.

Since the preliminary inhibitor experiments described above, indicatingdifference in K_(i) between the xylanases studied. The real K_(i) forseveral xylanases were determined. As can be seen from the data in FIG.22, the K_(i) values do differ significantly between the xylanases. Thisconfirms the results indicated by the simple preliminary inhibitorcharacterisation.

Inhibition as a Function of pH

A simple spot for xylanase inhibitor at a different pH revealed thatthere seemed to be an effect of pH on the inhibition of the xylanases.Therefore, an experiment was set up to examine this effect. As can beseen from FIG. 23 the inhibition of the xylanases are influenced by pH.FIG. 24 illustrates the pH optima for the xylanases. If these two curvesare compared, we see the highest inhibition at the pH optimum for thexylanase, except for the pH 4 measurement of the Novo xylanase (980901).

To determine whether the inhibition ratios measured in the assaysreported here are relevant in the dough, some calculations can be made:Inhibitor extraction Gram flour: 6 ml water: 12 g flour/ml: 0.5 g flourin assay: 0.05 Xylanase solution TXU/ml: 12 TXU/ml in assay: 3Inhibitor:xylanase TXU/kg flour: 60000 in inhibitor assay ratios TXU/kgflour: 3000 in bakery applications

From the above calculations, the inhibitor xylanase ratio in the assaycan be calculated to be 20 times lower in the assay than in dough. Thiscan only mean that the xylanase must be much more inhibited in dough.However, the mobility and water activity is much lower in dough and thismight influence the inhibition.

Summary Discussion

Wheat flour contains endogeneous endo-β-1,4-xylanase inhibitor. Theinhibitor can be extracted from wheat flour by a simple extraction usingwater, meaning that the inhibitor is water soluble. The inhibitor waspurified using gel filtration-, ion exchange- and hydrophobicinteraction chromatographic techniques.

Characterisation of the purified inhibitor, using analytical gelfiltration chromatography, SDS PAGE, reverse phase chromatography andMS, revealed a poly-peptide of approx. 40 KDa. This poly-peptide turnedout to be a di-peptide, containing two peptides with molecular weightsof 12104 and 28222 Da, respectively. The purified inhibitor (moreprecise the two peptides) was N-terminal sequenced, followed bydigestion and sequencing of peptides obtained.

The preliminary experiment with the inhibitor indicated that thedecrease in xylanase activity found could be due to proteolysis.However, analysis of incubation trials (xylanase+inhibitor) and kineticson the inhibitor indicated that the observed decrease in xylanaseactivity was due to a competitive inhibitor.

Inhibitor experiments using several xylanases indicate differences insensibility towards the inhibitor. Some xylanases are inhibited almost100% by the inhibitor (at a lower inhibitor xylanase ratio than presentin the flour). By varying pH in the inhibitor assay it turns out theinhibition is highly dependent on the pH in the assay. Examining thexylanase mutants revealed that changing one amino acid can mean a 250%decrease in inhibition.

To confirm the results described above, K_(i) values were determined forseveral xylanases. The results showed different K_(i)'s depending on thexylanase used, confirming the differences in resistance towards theinhibitor as function of xylanase seen in preliminary results.

Example 3

Baking Trials

The data below are from a baking trail with the XM1 mutant. The datashow that this novel xylanase mutant is clearly superior to BX (Bacillussubtilis wild type) based on volume. Based on stickiness measurementthere are no significant diffence between the two xylanases

Enzymes

980902 (BX):Purified Bacillus sub. wild type xylanase expressed in E.coli. (2000 TXU/ml)

980903 (XM1): Purified mutant of Bacillus sub. wild type xylanaseexpressed in E. coli. (1375 TXU/ml)

Flour

Danish flour, batch 98022.

Baking Test (Hard Crust Rolls)

Flour 2000 g, dry yeast 40 g, sugar 32 g, salt 32 g, GRINDSTED™ PanodanA2020 4 g, water 400 Brabender Units +4% were kneaded in a Hobart mixerwith hook for 2 minutes low speed and 9 minutes high speed. The doughtemperature was 26° C. The dough was scaled to 1350 gram. Resting 10minutes at 30° C. followed by moulding on a Fortuna moulder. Proofing 45minutes at 34° C., 85 % RH. Baked in a Bago-oven 18 minutes 220° C. andsteamed 12 seconds.

After cooling the rolls were scaled and their volume measured by therape seed deplacement method.${{Specific}\quad{volume}} = \frac{{{volume}\quad{of}\quad{the}\quad{bread}},{ml}}{{{weight}\quad{of}\quad{the}\quad{bread}},g}$Stickiness Measurement

Stickiness measurement was performed according to Protocol 2.

As can be seen from Table 15 the novel xylanase mutant (XM1) gives riseto significant higher bread volume increase than BX. TABLE 15 Breadvolume increase (ml/gram) and stickiness (g × s) as function of twoxylanases (BX and XM1) applied at different dosages. Dose, Stickiness,Specific Spec. vol. Sample TXU/kg g × s vol., ml/g increase, % BX 20006.00 6.03 2.55 BX 5000 6.60 6.49 10.37 BX 8000 5.00 6.77 15.14 BX 120007.00 6.72 14.29 XM1 2000 4.30 6.60 12.24 XM1 5000 6.20 6.88 17.01 XM18000 6.20 7.06 20.07 XM1 12000 6.90 7.32 24.49 Control 0 4.50 5.88 —

The data are shown in FIGS. 25, 26 and 27.

Example 4

Dough Stickiness as a Function of XM1, the Röhm Veron Special Xylanaseand a Purified Version of the Röhm Veron Special Xylanase

To determine whether the novel xylanase, XM1 gives more or less stickydough than Röhm's Veron Special xylanase (and a purified version herof)dough were prepared and stickiness as function of xylanase wasdetermined.

Flour

Danish flour, batch 98022 was used.

Dough Preparation

Dough were prepared as described in Protocol 2. After mixing the doughrested for 10 and 45 minutes, respectively, in sealed containers beforestickiness measurement.

Stickiness Measurement

Stickiness measurements were performed according to Protocol 2.

Enzymes

980903 (XM1):Purified mutant of Bacillus sub. wild type xylanaseexpressed in E. coli. (1375 TXU/ml)

#2199:the Röhm Veron Special xylanase (10500 TXU/g)

980603 (Röhm):Purified preparation of Frimond's Belase-xylanase(identical to Röhm's) (1050 TXU/ml)

The following doughs were made (Table 16): TABLE 16 Dough made fordetermination of stickiness Dosage, TXU/kg Xylanase flour 980603(Purified Röhm xylanase) 15.000 Control 0 XM1 15.000 #2199 (Röhm's VeronSpecial) 15.000

The dough in Table 16 gave the stickiness results in Table 17. TABLE 17Results from stickiness measurements on dough prepared with PurifiedRöhm xylanase, control, XM1 and the Röhm Veron Special xylanase.Stickiness Leavening Stickiness, increase, Xylanase TXU/kg flour time,min. g × s g × s 980603 15.000 10 7.22 2.22 980603 15.000 45 10.15 4.08Control 0 10 5.00 0 Control 0 45 6.09 0 XM1 15.000 10 6.61 1.61 XM115.000 45 9.64 3.55 #2199 15.000 10 8.57 3.57 #2199 15.000 45 12.14 6.05

The data are shown in FIG. 28, 29 and 30.

The increase in stickiness using the XM1 is lower than the stickinessincrease with the purified Röhm xylanase. The stickiness increaseobtained using the unpurified Röhm xylanase is much higher.

Example 5

Dough Stickiness as a Function of Bacterial Endo-β-1,4-Glucanase

The results in the following are from an experiment designed to studythe ability of bacterial Endo-β-1,4-Glucanase to give stickiness.

Enzymes

981102-1 (Xyl):Correspond to a purified preparation of Röhm's bacterialxylanase from the product Veron Special. The preparation is purexylanase and do not contain any Endo-β-1,4-Glucanase (350 TXU/ml)

981102-2 (Xyl+Gluc):Correspond to a purified preparation of Röhm'sbacterial xylanase from the product Veron Special, containingEndo-β-1,4-Glucanase (900 TXU/ml +19 BGU/ml)

Xylanase Assay

Xylanase assays were performed according to Protocol 1

Glucanase Assay

Glucanase assays were performed according to Protocol 4

Flour

Danish flour, batch no 98058 was used. The water absorbtions, at 400 BUis 60%.

Dough Preparation

Dough were prepared as described in Protocol 2. After mixing the doughrested for 10 and 45 minutes respectively at 30° C. in sealedcontainers.

Stickiness Measurement

Stickiness measurements were performed according to Protocol 2

The dough listed in Table 18 were prepared and examined for stickiness.TABLE 18 Dough prepared for examining stickiness Dough No. Dough TXU/kgflour BGU/kg flour 1 Control 0 0 2 TXU 7500 0 3 TXU + BGU 7500 158 4 TXU15000 0 5 TXU + BGU 15000 316

The dough listed in Table 18 gave the stickiness results in Table 19.TABLE 19 Stickiness results from dough with xylanase and xylanase +glucanase Dough No. refers to the dough No. in Table 18 Stik_10 indicateresults from stickiness measurements after 10 minutes Stik_45 indicatemeasurements after 45 minutes of resting Dough No. Stik_10, g × sstd.dev Stik_45, g × s std.dev 1 4.5 0.342 5.11 0.552 2 5.29 0.619 8.620.607 3 5.47 0.663 9.38 0.832 4 8.61 0.408 9.15 0.418 5 8.73 0.35 10.190.857

As can be seen from Table 191 the Endo-β-1,4-Glucanase addition to thedough increases the stickiness of the dough. The results from Table 19are illustrated in FIG. 31.

SUMMARY

In summary the present invention provides and the Examples show interalia:

-   -   a. The isolation of an endogenous endo-β-1,4-xylanase inhibitor        from wheat flour.    -   b. The characterisation of an endogenous endo-β-1,4-xylanase        inhibitor isolated from wheat flour.    -   c. The characterisation of the effect of endogenous        endo-β-1,4-xylanase inhibitor on different xylanases.    -   d. A means for selecting xylanases not detrimentally affected by        endogenous endo-β-1,4-xylanase inhibitor.    -   e. A means for selecting xylanases which are not detrimentally        affected by endo-β-1,4-xylanase inhibitors.    -   f. Xylanases that provide dough exhibiting favourable volume and        acceptable stickiness than when compared to doughs comprising        fungal xylanases.    -   g. A method for screening xylanases and/or mutating the same        using an endogenous endo-β-1,4-xylanase inhibitor, and the use        of those xylanases or mutants thereof in the manufacture of        doughs.    -   h. A foodstuff prepared with the xylanases of the present        invention.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the present invention will be apparentto those skilled in the art without departing from the scope and spiritof the present invention. Although the present invention has beendescribed in connection with specific preferred embodiments, it shouldbe understood that the invention as claimed should not be unduly limitedto such specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in biochemistry and biotechnology or related fields areintended to be within the scope of the following claims.

1-43. (canceled)
 44. A xylanase comprising the amino acid sequencepresented as SEQ ID No. 5.